A typical volatile agent,  isoflurane , has a saturated  vapor pressure  of 32kPa (about 1/3 of an atmosphere). This means that the gas mixture leaving the vaporizing chamber has a  partial pressure  of isoflurane of 32kPa. At sea-level ( atmospheric pressure  is about 101kPa), this equates conveniently to a  concentration  of 32%. However, the output of the vaporizer is typically set at 1–2%, which means that only a very small proportion of the fresh gas needs to be diverted through the vaporizing chamber (this proportion is known as the splitting ratio). It can also be seen that a plenum vaporizer can only work one way round: if it is connected in reverse, much larger volumes of gas enter the vaporizing chamber, and therefore potentially toxic or lethal concentrations of vapor may be delivered. (Technically, although the dial of the vaporizer is calibrated in volume percent (e.g. 2%), what it actually delivers is a partial pressure of anesthetic agent (e.g. 2kPa)).


The performance of the plenum vaporizer depends extensively on the saturated vapor pressure of the volatile agent. This is unique to each agent, so it follows that each agent must only be used in its own specific vaporizer. Several safety systems, such as the Fraser-Sweatman system, have been devised so that filling a plenum vaporizer with the wrong agent is extremely difficult. A mixture of two agents in a vaporizer could result in unpredictable performance from the vaporizer.


Saturated vapor pressure for any one agent varies with temperature, and plenum vaporizers are designed to operate within a specific temperature range. They have several features designed to compensate for temperature changes (especially cooling by  evaporation ). They often have a metal jacket weighing about 5 kg, which equilibrates with the temperature in the room and provides a source of heat. In addition, the entrance to the vaporizing chamber is controlled by a  bimetallic strip , which admits more gas to the chamber as it cools, to compensate for the loss of efficiency of evaporation.


The first temperature-compensated plenum vaporizer was the Cyprane 'FluoTEC'  Halothane  vaporizer, released onto the market shortly after Halothane was introduced into clinical practice in 1956.


The drawover vaporizer is driven by  negative pressure  developed by the patient, and must therefore have a low resistance to gas flow. Its performance depends on the  minute volume  of the patient: its output drops with increasing minute ventilation.


The design of the drawover vaporizer is much simpler: in general it is a simple glass reservoir mounted in the breathing attachment. Drawover vaporizers may be used with any liquid volatile agent (including older agents such as  diethyl ether  or  chloroform , although it would be dangerous to use  desflurane ). Because the performance of the vaporizer is so variable, accurate calibration is impossible. However, many designs have a lever which adjusts the amount of fresh gas which enters the vaporizing chamber.


The drawover vaporizer may be mounted either way round, and may be used in circuits where  re-breathing  takes place, or inside the circle breathing attachment.


Drawover vaporizers typically have no temperature compensating features. With prolonged use, the liquid agent may cool to the point where condensation and even frost may form on the outside of the reservoir. This cooling impairs the efficiency of the vaporizer. One way of minimising this effect is to place the vaporizer in a bowl of water.


The relative inefficiency of the drawover vaporizer contributes to its safety. A more efficient design would produce too much anesthetic vapor. The output concentration from a drawover vaporizer may greatly exceed that produced by a plenum vaporizer, especially at low flows. For safest use, the concentration of anesthetic vapor in the breathing attachment should be continuously monitored.


Despite its drawbacks, the drawover vaporizer is cheap to manufacture and easy to use. In addition, its portable design means that it can be used in the field or in  veterinary anesthesia .


The third category of vaporizer (the dual-circuit gas–vapor blender) was created specifically for the agent  desflurane . [ 5 ]  Desflurane boils at 23.5 °C, which is very close to room temperature. This means that at normal  operating temperatures , the saturated vapor pressure of desflurane changes greatly with only small fluctuations in temperature. This means that the features of a normal plenum vaporizer are not sufficient to ensure an accurate concentration of desflurane. Additionally, on a very warm day, all the desflurane would boil, and very high (potentially lethal) concentrations of desflurane might reach the patient.


A desflurane vaporizer (e.g. the TEC 6 produced by  Datex-Ohmeda ) is heated to 39C and pressurized to 194kPa. [ 6 ]  It is mounted on the anesthetic machine in the same way as a plenum vaporizer, but its function is quite different. It evaporates a chamber containing desflurane using heat, and injects small amounts of pure desflurane vapor into the fresh gas flow. A  transducer  senses the fresh gas flow. [ 5 ]


A warm-up period is required after switching on. The desflurane vaporizer will fail if mains power is lost. Alarms sound if the vaporizer is nearly empty. An electronic display indicates the level of desflurane in the vaporizer.


The expense and complexity of the desflurane vaporizer have contributed to the relative lack of popularity of desflurane, although in recent years it is gaining in popularity.


Historically,  ether  (the first volatile agent) was first used by  John Snow 's inhaler (1847) but was superseded by the use of  chloroform  (1848). Ether then slowly made a revival (1862–1872) with regular use via  Curt Schimmelbusch 's "mask", a  narcosis  mask for dripping liquid ether. Now obsolete, it was a mask constructed of wire, and covered with cloth.


Pressure and demand from  dental surgeons  for a more reliable method of administering ether helped modernize its delivery. In 1877,  Clover  invented an ether inhaler with a water jacket, and by the late 1899 alternatives to ether came to the fore, mainly due to the introduction of spinal anesthesia. Subsequently, this resulted in the decline of ether (1930–1956) use due to the introduction of  cyclopropane ,  trichloroethylene , and  halothane . By the 1980s, the anesthetic vaporizer had evolved considerably; subsequent modifications lead to a raft of additional safety features such as temperature compensation, a  bimetallic strip , temperature-adjusted splitting ratio and anti-spill measures.


The  breathing circuit  is the ducting through which the breathing gases flow from the machine to the patient and back, and includes components for mixing, adjusting, and monitoring the composition of the breathing gas, and for removing carbon dioxide.


A modern anaesthetic machine includes at minimum the following components: [ 2 ]


Systems for monitoring the patient's  heart rate ,  ECG ,  blood pressure  and  oxygen saturation  may be incorporated, in some cases with additional options for monitoring  end-tidal carbon dioxide  and  temperature . [ 2 ]   Breathing systems  are also typically incorporated, including a manual  reservoir bag  for ventilation in combination with an  adjustable pressure-limiting valve , as well as an integrated mechanical ventilator, to accurately ventilate the patient during anaesthesia. [ 2 ]


Based on experience gained from analysis of mishaps, the modern anaesthetic machine incorporates several safety devices, including:


The functions of the machine should be checked at the beginning of every operating list in a "cockpit-drill". Machines and associated equipment must be maintained and serviced regularly.


Older machines may lack some of the safety features and refinements present on newer machines. However, they were designed to be operated without  mains electricity , using compressed gas power for the ventilator and suction apparatus. Modern machines often have  battery  backup, but may fail when this becomes depleted.


The modern anaesthetic machine still retains all the key working principles of the Boyle's machine (a  British Oxygen Company  trade name) in honour of the British anaesthetist Henry Boyle. In India, however, the trade name 'Boyle' is registered with Boyle HealthCare Pvt. Ltd., Indore MP.


Various regulatory and professional bodies have formulated checklists for different countries. [ 8 ]  Machines should be cleaned between cases as they are at considerable risk of contamination with  pathogens . [ 9 ]


The general term  nebulizer  refers to an apparatus that converts liquids into a fine mist. Nozzles also convert liquids into a fine mist, but do so by pressure through small holes. Nebulizers generally use gas flows to deliver the mist. The most common form of nebulizers are medical appliances such as  asthma inhalers  or paint spray cans.  Analytical nebulizers  are a special category in that their purpose is to deliver a fine mist to spectrometric instruments for elemental analysis. They are necessary parts of  inductively coupled plasma atomic emission spectroscopy  ( ICP-AES ),  inductively coupled plasma mass spectrometry  (ICP-MS), and  atomic absorption spectroscopy  (AAS).


Analytical nebulizers are used in trace element analysis. This type of work plays an important role in areas of pharmaceutical and clinical study, biological, environmental and agricultural assessment and petroleum testing. They also have nuclear applications.


Most analytical pneumatic nebulizers use the same essential principle ( induction ) to  atomize  the liquid: When gas at a higher pressure exits from a small hole (the orifice) into gas at a lower pressure, it forms a gas jet into the lower pressure zone, and pushes the lower pressure gas away from the orifice. This creates a current in the lower pressure gas zone, and draws some of the lower pressure gas into the higher pressure gas jet. At the orifice, the draw of the lower pressure gas creates considerable suction, the extent depending on the differential pressures, the size of the orifice, and the shape of the orifice and surrounding apparatus. In all pneumatic induction nebulizers, the suction near the orifice is utilized to draw the liquid into the gas jet. The liquid is broken into small droplets in the process.


Present induction pneumatic nebulizer designs fit into 5 categories: 1. Concentric: Liquid flow surrounded by a Gas flow or Gas flow surrounded by a Liquid flow; 2. Cross Flow: Gas flow at right angles to the Liquid flow; 3. Entrained: Gas and Liquid mixed in the system and emitted as a combined flow. 4. Babington and V Groove: Liquid is spread over a surface to decrease the surface tension, and passed over a gas orifice; 5. Parallel Path: Liquid is delivered beside a gas orifice and induction pulls the liquid into the gas stream.


Newer non-induction nebulizers include 3 more categories: 6. Enhanced Parallel Path: Liquid is delivered beside a gas orifice and drawn into the gas stream by surface tension along a spout; 7: Flow Blurring: liquid is injected by pressure into a gas stream; 8. Vibrating Mesh: liquid is pushed through tiny holes by a vibrating ultrasonic plate.


Concentric nebulizers have a central capillary with the liquid and an outer capillary with the gas. The gas draws the liquid into the gas stream through induction, and the liquid is broken into a fine mist as it moves into the gas stream. In theory, the gas and liquid may be switched with the gas in the center and the liquid in the outer capillary, but generally they work better with the gas outside and the liquid inside. [ 1 ]  The first Canadian concentric patent was Canadian Patent #2405 of April 18, 1873. It was designed to deliver a better spray of oil into a burner. The design is larger but essentially the same as modern analytical nebulizers. The first one developed for spectrometers was a glass design developed by Dr. Meinhard of California in 1973. [ 2 ]  His design enabled early ICP users to have a consistent sample introduction nebulizer, but it plugged easily. Today many companies produce glass concentrics, and since 1997, Teflon concentrics have become available.


Cross flow nebulizers have a gas capillary set at right angles to the liquid capillary. The gas is blown across the liquid capillary and this produces a low pressure that draws the liquid into the gas stream. Generally the suction is similar to what is produced in a concentric nebulizer. The benefit of a cross flow is that the liquid capillary have a larger inside diameter allowing for more particles to pass through without plugging the nebulizer. The disadvantage is that the mist is usually not as fine or as consistent. [ 3 ]


There are no analytical nebulizers at present using this technique, but some oil burners do. Mainly used in much older designs as newer concentrics and cross flows are much better and easier to make.


V Groove nebulizers are similar to a cross flow in that the liquid is delivered in a capillary at right angles to the gas capillary, but the liquid is poured down a vertically orientated groove that flows past a gas orifice. The gas pulls the liquid into the gas flow and forms a fine mist. These allow for very large ID liquid capillaries, but have no suction and require a pump to feed the liquid to the device. They must be correctly orientated or they do not allow the liquid to flow past the gas stream. And their mist usually produces larger droplets than with concentrics or cross flows.


This design was developed by John Burgener of Burgener Research Inc. Here, the gas stream and sample run through the nebulizer in parallel capillaries. At the tip of the nebulizer, the liquid is pulled into the gas stream and then dispersed into the chamber as a mist.


This design was developed by John Burgener of Burgener Research Inc. [ 4 ]  Here, the gas stream and sample run through the nebulizer in parallel capillaries. At the tip of the nebulizer, the liquid is pulled into the gas stream by surface tension along a spout dipping into the gas stream. This allows the gas to impact the liquid, and has the liquid interact in the center of the gas flow where the gas flow speed is highest, producing a better transfer of energy from the gas to the liquid, and producing a finer droplet size. The Burgener Mira Mist nebulizers are the main products using the Enhanced Parallel Path method.


This is a new type of nebulizer which does not use induction to mix the sample and gas. Instead, pneumatic atomization is employed here, which results in the micro-mixing of fluids using a reflux cell. [ 5 ]  This means that there is a turbulent mixing of the liquid and gas which results in great sensitivity and is very efficient. The OneNeb is the only example of this sort.


Since 2011, this variation on ultrasonic nebulizers has been available. There is a vibrating membrane which has micro holes in it. The sample enters through the back and is pushed through the holes as the membrane vibrates. This makes a fine mist with a droplet size proportional to the hole size. This method requires no gas flow, and is used in conjunction with a chamber. If the droplets are less than 5μm then they are too small to stick to the chamber walls and the chamber remains dry while 90–100% of the sample makes it to the torch.


The early history of medical nebulizers can be read  here . The development of analytical nebulizers since the introduction of the ICP / ICP-MS is seen below: [ 6 ]


1970s Adjustable Cross flow (US patent #4,344,574) [ 7 ]


1974 Meinhard Concentric


1978 V-groove (by Suddendorf and Boyer) (US Patent #4,206,160) [ 8 ]


1980 Pillar and Post (by Garbarino and Taylor)


1983 GMK Nebulizer: Glass Babington V-groove


1983 Meinhard C-type nebulizer


1983 Precision glassblowing (similar to Minehard A-type)


1983 Jarrell Ash (Thermo) Sapphire V-groove


1983 Meddings' MAK: glass fixed cross flow


1984 Meinhard K-type: recessed inner capillary


1984 Glass Expansion begins making ICP glassware


1985 Burgener-Legere – first commercial teflon nebulizer – V-groove – no adjustable parts


1986 Direct injection micro nebulizer by Fassel, Rice & Lawrence (US patent #4,575,609) [ 9 ]


1986 Hildebrand Grid nebulizer


Late 1980s Perkin Elmer Gem Tip cross flow


1988 CETAC Ultrasonic Nebs


1980s Cyclonic chambers


1987 Glass Expansion's first neb – the VeeSpray (ceramic V-groove)


1989 Glass Expansion first concentric – the Conikal (machined instead of glass blown)


1989 Noordermeer Glass V Groove (US patent #4,880,164) [ 10 ]


1992 Glass Expansion – non salting Sea Spray


1993 Modified Lichte Glass V-Groove


1993 Burgener BTF – first Parallel Path Neb (US patent #5,411,208) [ 11 ]


1994–1995 Main Burgener Parallel Path Nebs – BTS 50, BTN & T2002


Mid 1990s Perkin Elmer GemCone: Miniature V-Groove


With the introduction of the ICP-MS to the laboratory, the creation of micro nebulizers became a priority in order to deliver smaller amounts of sample at lower flow rates.


1993 The Meinhard HEN (high efficiency nebulizer) was produced which handled very low flow rates but salted and plugged easily as a result. (25 times less sample than a standard Meinhard)


1997 Cetac Microconcentric Nebulizer – first Teflon concentric 50, 100, 200 or 400 μL/min


1997 Meinhard Direct Injection HEN – (DIHEN) (US Patent #6,166,379) [ 12 ]


1999 Elemental Scientific – PFA Concentric Nebs 20, 50, 100 or 400 μL/min


1999 Burgener Micro 1: Parallel Path


2000 Burgener Micro 3: Parallel Path


2001 Burgener Mira Mist: First Enhanced Parallel Path Nebulizer (US patent #6,634,572) [ 13 ]


2004 Epond Typhoon: Glass Concentric


2005 Ingeniatrics OneNeb: Flow Blurring Technology


2010 Epond Lucida: Teflon Micro Concentric


2012 Burgener PFA 250: PFA Micro flow Enhanced Parallel Path Nebulizer


2010 – 2013 Meinhard and Glass Expansion: Significant improvements in attachments and designs of glass concentrics.


AptarGroup, Inc.,  also known as  Aptar , is a United States–based global manufacturer of consumer dispensing packaging and  drug delivery  devices. [ 7 ] [ 1 ]  The group has manufacturing operations in 18 countries. [ 8 ]


The company began as Werner Die & Stamping in Cary, Illinois, in 1946 and later incorporated as AptarGroup in 1992. [ 1 ] [ 9 ]  Aptar originally developed spray valves and pumps for consumer and household products. The company later began producing  nasal administration  and  pulmonary drug delivery  devices such as  nasal spray  systems and  metered-dose inhaler  valves. [ 9 ] [ 10 ] [ 11 ] [ 12 ]  Biotech and pharmaceutical companies use Aptar's different Unidose and Bidose devices for the single or two-shot intranasal delivery of different medicines. [ 13 ]


In 2016, Aptar announced that it provided the delivery system for Adapt Pharma's  Narcan . [ 14 ]  Narcan is a naloxone hydrochloride nasal spray used as an emergency treatment for opioid overdoses. [ 14 ]  Aptar's liquid spray drug delivery technology platform works as a ready-to-use, single-shot, unit-dose system for Narcan. It was the first  FDA  approved nasally administered, ready-to-use medication used to reverse the effects of an opioid overdose. [ 14 ]  Narcan does not require any assembly, medical training, or needle injection. [ 15 ]


In 2016, Aptar entered into an agreement with  Becton Dickinson & Company  to develop new self-injection devices. [ 16 ] [ 17 ]


Aptar entered into an agreement in 2016 with Propeller Health Partners to develop a digitally connected medication  inhaler . [ 18 ]  The company made an investment in Propeller Health Partners (now part of  Resmed ) in 2018. [ 19 ]


In July 2019, the FDA-approved Aptar Pharma's Unidose Powder System as the first intranasally-delivered, needle-free rescue treatment for severe  hypoglycemia . [ 13 ]


In 2020, during the  COVID-19 pandemic , Aptar invested in new tools to accelerate its molding equipment and assembly machines for pumps, but it still wasn't enough to keep up with demand. [ 20 ]


In 2012, Aptar acquired Stelmi, a manufacturer of  elastomer  primary packaging components. [ 21 ]  In 2016, Aptar acquired Mega Airless, a manufacturer of airless packaging solutions. [ 22 ]  In 2018, Aptar acquired CSP Technologies, a material science company that manufactures  active packaging  solutions. [ 23 ]


In June 2019, Aptar acquired two companies, Nanopharm and Gateway Analytical. [ 24 ]  In November 2019, the company acquired Noble International, which specializes in training devices and patient onboarding. [ 25 ]  In February 2020, Aptar acquired FusionPKG, a makeup packaging company. [ 26 ] [ 27 ]


In November 2020, the company acquired the digital respiratory health company Cohero Health. [ 28 ] [ 29 ]


In July 2021 Aptar acquired the digital therapeutics company, Voluntis (ENXTPA: ALVTX), and 80% of the equity interests of Weihai Hengyu Medical Products Co., Ltd., a Chinese manufacturer of elastomeric and plastic components used in injectable drug delivery. [ 30 ]


Aptar was named to  Barron's  list of the Top 100 Most Sustainable U.S. Companies in 2019, 2020, 2021, and 2022. [ 31 ] [ 32 ] [ 33 ] [ 34 ]  At the end of 2020, 85% of the company’s global electricity use came from renewable sources. [ 35 ]  It was also named by  Newsweek  as one of America's Most Responsible Companies in 2021, [ 36 ]  2022, [ 37 ]  and 2023 [ 38 ]  and received an A score for climate change from the  Climate Disclosure Project . [ 39 ]


In September 2019, the company announced a partnership with Loop, a shopping platform from  TerraCycle  that delivers products in reusable containers. [ 40 ]  The company made the  Forbes  Green Growth 50 List in 2021. [ 41 ]


Aquasomes  are self-assembling  nanoparticle  drug carrier systems composed of three layers: a  ceramic  core, an  oligomer  coat, and a loaded  biochemically active molecule . Aquasomes are utilized for targeted  drug delivery  to achieve specific therapeutic effects, and are  biocompatible ,  biodegradable , and stable. Due to their structure, aquasomes are capable of delivering several types of substrates, and can be used for applications such as delivery of  antigens ,  insulin , and  hemoglobin .


Aquasomes were first investigated by Kossovsky et al. in 1996 in experiments proposing their use in antigen delivery, drug delivery, and hemoglobin delivery systems. [ 2 ]  This initial research described aquasomes as self-assembling, with a novel surface modification process allowing the immobilization of drugs on the surface. [ 2 ]  The research was intended to address the molecular denaturation of  polypeptide  pharmaceuticals. [ 2 ]  Kossovsky et al. suggested that this system would be able to combat physical and chemical degradative agents affecting bioactive molecules while preserving the molecular structure of the drug. [ 2 ]


Since this initial exploration, the understanding of the composition and applications of aquasomes has increased. After each individual layer is synthesized, aquasomes self-assemble into triple-layered particles. The tri-layer structure enables aquasomes to deliver and release poorly soluble drugs in a controlled manner. Delivery of these poorly soluble drugs within aquasomes increases their solubility, bioavailability, and stability. These drugs are  adsorbed  onto the surface of the aquasome, forming its third layer, which confers bioactive properties to the aquasome.


Aquasomes form a three-layered structure, made of a  polyhydroxy   oligomer  coated core upon which the drug is loaded. The biochemically active molecules are able to interact with the coated core through different  Van der Waal forces ,  entropic forces , and  ionic  and  non-covalent bonds . [ 3 ]  The structure of aquasomes enables them to carry a variety of substrates (chemicals), facilitating applications such as protein and  peptide  delivery and protection, and the delivery of  nucleic acids  for  gene therapy  applications. [ 1 ]


Aquasomes’ solid core, made of ceramic or  polymeric  material, is attributed to the structural stability of the nanoparticle itself, and can result in improved solubility and  biocompatibility  of the drug. [ 1 ]  Different core designs have also been shown to affect the controlled release properties of the drug molecule. A commonly used core material is the ceramic  calcium phosphate , which naturally occurs in the body. [ 1 ]   Hydroxyapatite , which is found in bone, is another commonly used core material. Hydroxyapatite cores have been shown to contribute to targeted delivery of encapsulated  hepatitis B  antigens intracellularly. [ 1 ]


The second layer of aquasomes is the  carbohydrate  coat, onto which the drug is adsorbed. Due to carbohydrate’s action as a dehydroprotectant, [ definition needed ]  it has been shown to function as a natural stabilizer to preserve the conformation (shape) of  soft drugs . [ 1 ]  The dehydroprotectant property of the carbohydrate coat also enables protection of the biochemically active molecule from  dehydration  and  protein degradation . [ 1 ]


The size of aquasomes ranges from 60 to 300 nanometers, hence their characterization as a nanoparticle drug carrier. [ 2 ]  The nanoscale of aquasomes gives them a high surface area to volume ratio. The smaller the core, the higher the surface area to volume ratio, which increases the drug loading capacity of the aquasome. [ 3 ]  Aquasomes possess water-like properties due to the presence of the carbohydrate coating, enabling them to protect and preserve fragile biological molecules. The size of aquasome particles increases as a function of the ratio between the concentration of the core to the coat due to the availability of free surface core particles for the coating material. [ 1 ]


The  self-assembly  process of aquasomes into their tri-layer structure is achieved by non-covalent and ionic bonds, along with physicochemical properties of their components. Calcium phosphate nanoparticles are formed before the carbohydrate coat is  adsorbed  onto the surface of the core through  electrostatic interactions . Layers are then added to the structure to achieve desired size, while  crosslinked  polymers aid in further stabilization. The  sonication  process during the reaction of  disodium hydrogen phosphate  and  calcium chloride  to prepare calcium phosphate impacts the self-assembly process of aquasomes by increasing  surface free energy  of the core prepared. This assembly process allows the design of aquasomes for specific drug delivery applications. [ 3 ]


The structure of aquasomes can contribute to controlled drug release, drug stability, and intracellular targeting of the drug. Other commonly used nanoparticle drug delivery systems include  niosomes ,  liposomes , and  vesosomes , the compositions of which contribute to different properties of the resulting nanoparticle compared to aquasomes. Niosomes are composed of non-ionic  surfactants  and bilayer structures, allowing them to encapsulate  hydrophilic  and  hydrophobic  drugs. Liposomes are composed of  phospholipids  and a similar bilayer structure to niosomes, and can deliver toxic or poorly soluble drugs. Vesosomes have a core-shell structure similar to aquasomes, but contain a lipid bilayer core and a polymer shell, while aquasomes consist of a ceramic or polymeric core and a carbohydrate coat. Vesosomes are used for encapsulating  imaging agents  and aiding in imaging techniques such as  MRI .  [ 4 ] [ 5 ]


The three major units of aquasomes are fabricated together according to  self-assembly , a thermodynamically driven process that organizes subunits of a system in a manner that results in the lowest  Gibbs free energy  available, known as ΔG. Self-assembly as a mixing process offers high accuracy and control over sizes on the nanometer scale, which is especially relevant for aquasomes, which exist on this size scale. The three layers of aquasomes can be synthesized differently using a variety of techniques depending on the intended functions or desired therapeutic effects. [ 1 ]  The general scheme of aquasome fabrication involves a sequential synthesis of a  nanocrystalline  core, followed by a  polyhydroxy  coating, and finished with integration of bioactive molecules. Throughout this process, several intermittent steps are included that involve selective filtering and purification to remove byproducts while isolating the desired products for further processing. [ 1 ]


The core of an aquasome can be made from either ceramic or  polymeric  materials. Examples of such polymers include  acrylates  and  gelatin . However, because ceramic materials are more ordered due to their naturally occurring crystalline structure, they are more often preferred as the material type for the core. [ 1 ]  Some of the most common ceramic materials used in the formation of an aquasome core include  tin oxide ,  calcium phosphate , and even  diamond . Another characteristic that ceramic materials provide is enhanced binding of the carbohydrate layer due to the high  surface energy  present on the orderly surface. The binding affinity of the carbohydrate layer also reduces  surface tension  for its bond to the ceramic core. [ 2 ]  The first aquasomes fabricated with a nanocrystalline core using ceramic material are detailed in Kossovsky et al. in 1996. [ 2 ]  Calcium phosphate ceramic nanoparticles ( brushite ) were first prepared via the method of solution precipitation and  sonication . [ 2 ]  Precipitation methods are the most common techniques employed when synthesizing the core of an aquasome as they offer control over the homogeneity and purity of the precipitated products, which are important design features in the core structure. [ 1 ]  Once the cores are prepared, they are separated by  centrifugation  and then washed to remove any  salt  byproducts from the solution precipitation process. Finally, the washed cores are passed through a  Millipore filter  to selectively isolate core particles of a certain size. [ 2 ]


After synthesizing and purifying the core, the carbohydrate layer is added to its surface. Common coating materials are typically  polyhydroxy   oligomers  such as  cellobiose ,  citrate ,  lactose , and  sucrose . [ 2 ]  This layer seems to be important for the properties of aquasomes, as it influences several drug characteristics including adsorption, molecular stability, and conformation (shape), and acts as a dehydroprotectant. [ 1 ] [ definition needed ]  The addition of the carbohydrate layer to the surface of the nanocrystalline core is commonly carried out by passive adsorption through incubation [ clarification needed ]  and  sonication . Similar to the processing of the core, the carbohydrate layer is subjected to centrifugation, washing, and further sonification followed by heated air drying. [ 1 ]


Finally, the bioactive molecule of interest is loaded into the carbohydrate layer. This process typically occurs through either  lyophilization  or passive adsorption, and the fully functionalized aquasome is then characterized. [ 1 ]


Solution precipitation as a core synthesis technique produces homogenous-sized nanoparticles, which can be advantageous in controlling specific physical properties such as  surface tension  and  packing density  of the atoms in a  crystalline lattice  structure. [ 1 ]  The most common methods of characterizing nanoparticle size distribution and morphology of the core in aquasomes include  scanning electron microscopy  (SEM) and  transmission electron microscopy  (TEM). [ citation needed ]  In a study by Kommimeni et al. in 2012, researchers employed TEM to verify that the ceramic particles were spherical and also in the acceptable nano-range for aquasomes. [ 6 ]  The carbohydrate coating size can also be characterized using SEM and TEM, but  Fourier-transform infrared spectroscopy  (FTIR) is commonly utilized to check for the presence of the coat. [ citation needed ]  In a study by Kommimeni et al. in 2020, FTIR was used to confirm the presence of the coating by analyzing the IR spectra bands that correspond to the functional groups of either the core or the sugar coat. [ 6 ]


The bioactive drug loaded onto the aquasome can be characterized in a variety of ways depending on the molecular classification of the drug. In Kossovsky et al. in 1996, which studied the effect of insulin as the bioactive drug of interest, immunogold labeling was employed. Through this technique, the different binding efficiencies of carbohydrate coatings for insulin were able to be observed. [ 2 ]


The structure of aquasomes enables dual drug delivery, or the delivery of two drugs simultaneously. This practice aims to enhance the therapeutic efficiency and reduce the side effects of the drugs delivered. Such systems can be useful in treating patients suffering from multiple diseases. Challenges in dual drug delivery include independently controlling release rates of each of the drugs loaded in the system. In a 2019 study by Damera et al., aquasomes were used to deliver  bovine serum albumin  (BSA) in combination with one of three therapeutic drugs ( Coumarin 153 ,  Warfarin , and  Ibuprofen ), allowing release of a  bioactive  molecule and a  hydrophobic  drug simultaneously. [ 7 ]  Damera et al. suggested that dual drug delivery was enabled by the bioactive molecule layer of the aquasome being BSA. This BSA layer interacted with the hydrophobic therapeutic drugs, and the strength of the binding interactions was shown to affect the release behaviors of the drugs. Dual drug delivery with aquasomes thus shows promise for treatment of patients with coexisting diseases alongside  hypoalbuminemia , as the albumin from BSA can treat the hypoalbuminemia while the additional drug treats the disease. [ 7 ]


Aquasomes have been explored as carriers for  hemoglobin  throughout the body. In a 2002 study by Khopade, Khopade, and Jain, aquasomes were used to act as red blood cell substitutes with hemoglobin attached to the  oligomer  surface. Aquasomes in this application demonstrated minimal toxicity while obtaining a hemoglobin content of 80%, supplying blood and oxygen in a manner similar to regular red blood cells. [ 8 ]  Hemoglobin aquasomes with spherical  hydroxyapatite  cores have been shown to retain oxygen- affinity  and  cooperativity  for 30 days in rats in vivo, causing no red blood cell  hemolysis  or  blood coagulation , demonstrating potential capability as effective oxygen transporters. Additionally, aquasomes protected hemoglobin from degradation while maintaining hemoglobin function. Future exploration of aquasomes as hemoglobin carriers may explore controlled release of the aquasomes themselves to mimic typical oxygen release properties to aid in biomedical applications that require specific targeting and delivery of hemoglobin. [ 8 ]


Aquasomes with  calcium phosphate  ceramic cores may be useful for the pharmaceutical administration of substrates such as  insulin  where drug action is  conformationally specific . In a 2000 study by Cherian et al.,  disaccharides  such as  trehalose  were used to coat the core before insulin was loaded onto the coated cores via  adsorption . [ 9 ]  Albino rats were used as test subjects to test these aquasome insulin formulations, and the efficiency of different carbohydrate coat molecules on the aquasome was explored.  Pyridoxal-5-phosphate -coated particles were shown to lower blood glucose levels more efficiently when compared to trehalose- or  cellobiose -coated particles, which may be due to their differences in structural stability. [ 9 ]  The use of these nanoparticles for the delivery of insulin in vivo in rabbits demonstrated that insulin-bearing aquasomes showed slower release and prolonged activity compared to standard insulin solution. [ 9 ]  Similar to their role in carrying hemoglobin, the carbohydrate layer of aquasomes may be responsible for the ability to protect insulin from degradation when injected subcutaneously as in the albino rats tested. Aquasomes were also shown to release insulin in controlled manners, mimicking the typical release of insulin from the  pancreas . [ 9 ]


A potential challenge of aquasome-based drug delivery could be toxicity due to burst release of drugs if poorly absorbed on the carbohydrate coat. [ 1 ]  Aquasomes can also be expensive to formulate, particularly due to their step-by-step synthesis. Careful attention is needed during aquasome production to tune the thickness of each layer, and  leaching  and  aggregation  may occur during prolonged storage of aquasomes. A physiological challenge aquasomes present is that upon their entry into the bloodstream, they may be taken up nonspecifically, [ definition needed ]  leading to  opsonization  and  phagocytic clearance  by the immune system. To prevent this, aquasome surfaces can be coated with  polyethylene glycol  (PEG) to block  opsonin  binding through  steric hindrance ; however, the effect of PEGylation on aquasome drug release has not been sufficiently explored to enable clinical applications. [ 1 ]   Polymer degradation  in different physiological environments can change the stability and drug loading of aquasomes over time, as their surface properties directly impact drug release. Aquasomes may also be challenging to scale up and prepare as it is difficult to ensure consistent formulation quality. More research is needed to demonstrate both the efficiency and safety of aquasomes in clinical use. [ 1 ]


Further advances in aquasome research require additional investigation of their  in vivo  drug release and targeting. Applications such as delivery of  dithranol  for the treatment of  psoriasis  and oral delivery of  bromelain  for the treatment of inflammatory diseases such as cancer show promising results  in vitro  and  ex vivo . However, such applications have been unexplored in vivo, limiting their clinical use. Applications using aquasomes as carriers of hemoglobin, vaccines, and insulin have been tested in vivo in small animal models such as rats, mice, and rabbits, but current literature lacks in vivo testing in more advanced animal models, preventing their use as treatments for human conditions. Aquasomes are promising drug delivery mechanisms due to their ability to stabilize and transport a variety of substrates while allowing for controlled drug release. Prior to expanding the clinical applications of aquasomes, the gap existing in current literature will need to be filled by further investigating immune clearance of aquasomes, exploring additional surface modifications such as PEGylation, and expanding in vivo drug testing. [ 1 ]


An  autoinjector  (or  auto-injector ) is a  medical device  for injection of a premeasured  dose  of a particular  drug . Most autoinjectors are one-use,  disposable ,  spring -loaded  syringes  ( prefilled syringes ). By design, autoinjectors are easy to use and are intended for self-administration by patients, administration by untrained personnel, or easy use by healthcare professionals; they can also overcome the hesitation associated with self-administration using a needle. [ 1 ]  The site of injection depends on the drug, but it typically is administered into the  thigh  or the  buttocks . [ citation needed ]


Autoinjectors are  sharps waste . [ citation needed ]


Automatic syringes are known since  the 1910s, [ 2 ]  and many spring-loaded devices with needle protectors were patented in the first half of the 20th century, [ 3 ]  but it was not until 1970s when they became economically feasible to mass-produce (simple  syrettes  were used instead before). In 2023 an  open source  autoinjector was developed that could be digitally replicated with a low cost desktop  3D printer . [ 4 ]  It was tested against the then current standard (ISO 11608–1:2022) [ 5 ]  for needle-based injection systems and found to cost less than mass manufactured systems. [ 4 ]


Designs exist for both  intramuscular  and  subcutaneous injection . Disposable autoinjectors commonly use a pre-loaded spring as a power source. This spring and the associated mechanical components form a one-shot  linear actuator . [ citation needed ]  When triggered the actuator drives a three-step sequence: [ citation needed ]


Some injectors are triggered by simply pushing the nose ring against the injection site. In these designs, the protective cap is the primary safety. Other designs use a safety mechanism similar to  nail guns : The injection is triggered by pushing the nose ring against the injection site and simultaneously, while applying pressure, pushing a trigger button at the rear end of the device. [ citation needed ]


Since spent autoinjectors contain a  hypodermic needle , they pose a potential biohazard to waste management workers. Hence the protective cap is designed not only to protect the drug and keep the needle  sterile  but also to provide adequate sharps waste confinement after disposal. [ citation needed ]


Injectors intended for application through layers of clothing may feature an adjustable injection depth. Other typical features include: A drug inspection window, a color-coded spent indicator, and an audible click after the injection has finished. [ citation needed ]


Military uses include:


Another design has a shape and size of a  smartphone  which can be put into a pocket. This design also has a retractable needle and automated voice instructions to assist the users on how to correctly use the autoinjector. The "Auvi-Q" epinephrine autoinjector uses this design. [ 8 ]


A newer variant of the autoinjector is the gas  jet autoinjector , which contains a cylinder of pressurized gas and propels a fine jet of liquid through the skin without using a needle. This has the advantage that patients who fear needles are more accepting of using these devices. The autoinjector can be reloaded, and various doses or different drugs can be used, although the only widespread application to date has been for the administration of  insulin  in the treatment of  diabetes . [ 9 ] [ 10 ]


The  bifurcated needle  is a narrow steel rod, approximately 5 cm (2 in) long with two prongs at one end. [ 1 ]  It was designed to hold one dose of reconstituted freeze-dried  smallpox vaccine  between its prongs. [ 2 ]  Up to one hundred vaccinations can be given from one vial of the reconstituted vaccine. The established technique for smallpox vaccination is to dip the needle in the vaccine, and then perpendicularly puncture a person's upper arm fifteen times rapidly in a small circular area. Though skin in the area should be clean, use of an alcohol swab is optional and if used the skin must be allowed to fully dry to avoid deactivation of the live virus vaccine. The punctures should remain in an area approximately 5 mm in diameter. The needle is then disposed of. When done correctly a trace of blood appears at the vaccination site within 10 to 20 seconds after the procedure. [ 3 ] [ 4 ]


The bifurcated needle was created as a more efficient and cost effective alternative to the  jet injector  previously in use. It was invented in 1965 by Dr.  Benjamin Rubin , working at Wyeth Laboratories, and was the primary instrument used during the  World Health Organization 's 1966–1977 campaign to  eradicate smallpox . [ 5 ]  Rubin estimated that it was used to administer 200 million vaccinations per year during the final years of the campaign. [ 2 ]


Bioprinting drug delivery  is a method for producing  drug delivery  vehicles. It uses  3D printing  of  biomaterials . Such vehicles are biocompatible, tissue-specific  hydrogels  or implantable devices. 3D bioprinting prints cells and biological  molecules  to form tissues, organs, or biological materials in a scaffold-free manner that mimics living human tissue. The technique allows targeted disease treatments with scalable and complex geometry.


This technique was first developed in the 1950s as patients with incurable diseases sought  organ transplantations  beyond those available from donors. Organ transplantation showed limitations with immune responses and organ rejection. [ 1 ]


Techniques that have been studied include bioprinting hydrogels with various  bio-ink  (cell-laden microgel) materials and bioprinting implantable devices that mimic specific tissues or biological functions. Applications include promoting wound healing by delivering  antibiotics , anti-inflammatory treatments, or drugs that promote  cell differentiation  and  cell proliferation , providing anticancer treatments directly to tumors, and promoting/inhibiting  angiogenesis  and  vascularization  to treat cancer, arterial diseases, heart diseases, and arthritis. In addition, implants can be printed in unique shapes and forms to deliver drugs directly to targeted tissues. One approach adds a fourth dimension, which allows the materials to conform, by folding/unfolding, to release drugs in a more controlled manner. Bioprinting allows for biocompatible, biodegradable, universal, and personalized delivery vehicles.


Layer-by-layer printing of biochemicals and living cells requires precise placement and viable materials. The basic technology of a bioprinter starts with data taken from  computer-assisted design  ( CAD ) or a similar program, uses motion control systems to control the X/Y/Z axis direction drive mechanisms along with a material control system for the bio-ink printhead, and deposits material into a 3D construct. [ 2 ]  Bioprinting can be done by material jetting, material extrusion, or vat polymerization. [ 2 ] [ 3 ]


Material jetting, sometimes referred to as  fused deposition modeling  (FDM), is a method that involves depositing cells using  piezoelectric /thermal ink-jetting, acoustic wave jetting,  electrohydrodynamic jetting , or laser-induced forward transfer (LIFT). [ 3 ]  Piezoelectric/thermal ink-jetting uses the same non-contact process as desktop  inkjet printers  by pressuring material into a nozzle that expels droplets. Acoustic wave jetting uses acoustic radiation force to produce droplets; electrohydrodynamic jetting uses electric voltage to form droplets; and LIFT is replaces nozzles with a laser and generates a high-pressure bubble that propels droplets. [ 3 ]  These methods provide precise placement of the bioink and enable scaffold-free bioprinting.


Another method of bioprinting is extrusion. This is a mechanical method that uses motors to drive a piston. Extrusion is based on the rate of the motor's displacement, where the difference between the piston-driven pressure and  ambient pressure  drives the material through an angular turn of a rotary screw. [ 3 ]


Vat polymerization printing (VPP) uses a cell-hydrogel suspension. The constructs are formed layer-by-layer through laser curing in  stereolithography  (SLA) or UV  digital light processing  (DLP) into the vat of a photopolymer using a micromirror device. [ 3 ]


Hydrogels  are three-dimensional polymeric networks that can maintain their structure while absorbing large amounts of water or biological fluids. Hydrogels can be made of many different synthetic polymers or natural polysaccharides. These have been widely studied due to their similarities to the human  extracellular matrix  (ECM) and their ability to encapsulate drugs. They are mainly printed using jetting and extrusion. [ 3 ]


A common polysaccharide used in biomedical hydrogel applications is  alginate , a naturally occurring polyanionic  copolymer . [ 1 ]  The structure and high water absorption of alginate provides a tissue environment that closely mimics human soft tissue. In addition, it is an ideal candidate for biomedical applications due to its natural biodegradability and biocompatibility. This hydrogel leverages the delivery of drugs, protects drugs with encapsulation, and allows for tunable drug release and degradability


To construct alginate hydrogels, a series of negatively and positively charged polyelectrolytes are assembled layer-by-layer. Alginate is used as the matrix in bio-ink that is extruded from the bioprinter's syringe with increasing shear, resulting in a tough hydrogel with low  viscosity . Depending on the surrounding medium, alginate has the potential to form two different types of gels. Low-pH alginate shrinks and produces a viscous acidic gel, holding onto encapsulated drugs. Once the pH increases, such as inside an intestinal tract, alginate turns into a viscose gel that allows drug dissolution and release. This process allows for a controlled and sustained release to specific tissues.


One 2018 study used alginate-based hydrogels combined with the growth factor  platelet-rich plasma  (PRP) to develop a bio-ink with personalized biological factors. The plasms was extracted from specific patients, then mixed with the alginate solution. The solution was coated with  calcium chloride   agarose  gel. The result was a hydrogel disk that had decreased the risk of immune responses from the patient. The disk showed potential for promoting  mesenchymal stem cells  and  endothelial  cells in tissue healing. The study reported that PRP and alginate hydrogel bio-ink could be used by any bioprinter to produce personalized drug delivery therapies. [ 4 ]


The hydrogel can be loaded with any drug, and target any tissue. The low toxicity and controllable factors of alginate make it a suitable candidate for hydrogel incorporation. Alginate hydrogels have been used to deliver  bortezomib , an anticancer drug,  tetracycline hydrochloride  and  silver sulfadiazine , which are hydrophobic antibiotics, and  simvastatin , which promotes local stem cell differentiation. [ 5 ]


Another bio-ink is low molecular weight self-assembling  peptide -based materials. Peptide-based hydrogels are candidates for bio-inks since they resemble the ECM. In addition, their mechanical strength and stiffness of up to 40 kPa allow for strong and rigid hydrogels. [ 6 ]


A 2019 study used the "helical coiling of  micelles  induced by disulfide crosslinking in a lyotropic peptide  liquid crystal " to produce a printable hydrogel. They reported that the tripeptide self-assembled into a viscous solution of aligned micelles at high pH values that could be transformed into a self-supporting hydrogel when the cross-linking of the  sulfhydryl  group of the side chain peptides increased the  storage modulus  of the solution. [ 7 ]


These self-assembling peptides provide beneficial organization and strength. In addition, their resemblance to the native cellular microenvironment and tunable mechanical strength allow them to support the proliferation of human stem cells. Using self-assembling peptides to print hydrogels provides drug delivery vehicles that represent the ECM and potentially differentiate primary cells into organotypic structures and deliver antimicrobial, anti-inflammatory, anticancer, and wound healing drugs. [ 6 ] [ 7 ]  Specifically, hydrogels made of such peptides have been studied to encapsulate  chemotherapy  drugs that can disassemble and release the loaded drug under the stimulation of tumor environments, providing an alternative to typical chemotherapy that inevitably damages healthy cells, while killing cancer cells. [ 8 ]


Common protein-based bio-inks include  collagen ,  keratin ,  gelatin , and silk. These proteins are advantageous since they are in the ECM and display good cytocompatibility, biocompatibility, and biodegradability. They are all derived from natural sources, are isolated with different methods, and have various advantages and disadvantages. Collagen is typically printed using extrusion or SLA and provides good structural responses and esion for cells. [ 9 ] [ 10 ] [ 11 ]  Silk is printed using digital light processing, and provides strength and robustness. One disadvantage of silk is its potential to conform in response to high shear forces. [ 9 ] [ 10 ] [ 11 ] [ 12 ]  Gelatin is printed using extrusion and provides good cellular affinity, however, its covalent crosslinking-based stabilization requires chemical reactions that are not cytocompatible. [ 9 ] [ 10 ] [ 11 ] [ 12 ] [ 13 ]  Overall, protein-based bio-inks are abundant, inexpensive, biocompatible, and biodegradable, and are in common use for 3D bioprinting. Advantages of protein-based bio-inks over synthetic bio-inks include their similarity to human host tissue and their ability to match their degradation rate with the regeneration of host tissue.


Multiple protein applications use bio-inks for 3D printing. A 2014 study bioprinted cell-laden methacrylated gelatin (GelMA) hydrogels at concentrations ranging from 7 to 15% with varying cell densities. [ 14 ]  The study used "direct-write bioprinting of cell-laden photolabile ECM-derived hydrogels". [ 14 ]  They reported a direct correlation between printability and hydrogel mechanical properties. A commercially available bioprinter dispensed the GelMA hydrogel fibers using  digital light processing  since GelMA is  photosensitive . The hydrogels provided cell viability for at least eight days. [ 14 ]


Other research includes gelatin-sulfonated skin composite tissue to deliver cells to open wounds by seeding matrices. [ 15 ] [ 16 ]  Doing this helps wounds to heal faster and more efficiently. Gelatin hydrogels have successfully delivered  fluorescein , a hydrophobic molecule, and  microRNA  to promote  osteogenic  cell differentiation. [ 13 ]  Silk has successfully delivered  aspirin , an anti-inflammatory drug that aids in wound dressing, and  gentamicin , an antibiotic that also aids in wound dressing. [ 13 ]


Another bio-ink that has been successful in producing drug delivery systems via bioprinting is cellulosic nanomaterials.  Cellulose -based bio-inks are accessible, inexpensive, biodegradable, biocompatible, and stiff. A polysaccharide is obtained from the biosynthesis of plants and bacteria. It is extracted from raw materials with mechanical shearing actions and biological treatments, such as  hydrolysis , resulting in highly structured nanofibrils. Cellulose materials are defined by their high viscosity and shear-thinning behavior. [ 17 ]


One 2015 study used nanocellulose bio-inks as wound dressings. Extrusion produced porous structures with ionic  calcium chloride  cross-linking. These porous hydrogels were reported to support bacterial growth and incorporate and release antimicrobial drugs. These structures provide strong, moist environments that are ideal for delivering drugs to tissues that require wound healing aid. [ 18 ]


Non-hydrogel delivery systems implants are printed in the same manner as hydrogels. A 2022 study used SLA 3D printing to produce an implant to deliver drugs to the ear. 0.5%  levofloxacin  was added to a flexible resin. Mechanical and  in vitro  tests measured levofloxacin release. The results showed no interaction between the resin and the drug, the resistance of the implant without compromise, and high antimicrobial activity. Antibiotics were delivered directly to the inner ear to address infection. [ 19 ]


Researchers studied a printed bladder device for intravesical drug delivery. [ 20 ]  Intravesical instillation provides an alternative to oral medication and delivers high drug concentrations to specific sites. Studies reported the use indwelling bladder devices with an elastic polymer bio-ink to deliver  lidocaine hydrochloride  directly to the target site.


One study deposited perfusable vascular structures with a cell-responsive bio-ink that consisted of GelMA, sodium alginate, and poly(ethylene glycol)-tetra-acrylate (PEGTA). [ 21 ]  The study reported that this supported the spreading and proliferation of encapsulated endothelial and stem cells, leading to the formation of perfusable vessels. These may lead to the application of vascularized tissue constructs in organ transplantation and repair. Bioprinting vascular structures may lead to treatments for cancer, arterial disease, heart disease, and arthritis by regulating vascularization and  angiogenesis .


Cellulose nanofibrils have been used as a bio-ink for non-hydrogel applications. A 2017 study evaluated the use of bioprinting cellulose as drug-loaded implants. Researchers used FDM to evaluate drug release behavior. Fluorescent dye quinine was used to visualize the distribution of drugs in the implants. Quinine filaments were loaded into bio-printed cellulose implants and then incubated to observe their drug-release behaviors. The study reported showed that around 5% of the quinine was released from the cellulose implant over 100 days. Using cellulose nanofibrils might provide implants with customizable shapes and controlled release of loaded drugs via FDM. [ 22 ]


One study added a fourth dimension to the devices, which allows printed objects to change their shapes and functions as external factors are applied, broadening the range of biomedical applications as cellular self-organization becomes possible. [ 23 ]  This technique allows for more advanced control of drug release. Studies have reported results in the use of responsive materials and bio-inks. Responsive materials can reshape in response to stimuli, such as transforming via self-folding, assembling, and disassembling. Certain bio-inks have been reported to undergo maturation with cellular coating, self-organization, and matrix deposition.


Examples include a self-folding hydrogel  in vitro  model for  ductal carcinoma . [ 24 ]  The study attempted to create a self-folding curved hydrogel microstructure to mimic the geometry of ducts and  acini  within  mammary glands . The researchers used microstructures composed of poly(ethylene glycol)-diacrylate (PEGDA) and copolymerized the PEGDA to synthesize microstructures with increased cell adherence. Curved and tubular structures were fabricated via bioprinting, and the proliferation of cells on the outer surface, along with encapsulation of cells on the inner surface, was observed.


4D printing with thermally actuating hydrogels was reported to be relatively fast and reversible with skeletal muscle-like linear actuation in tough hydrogel materials that control the flow of water. [ 25 ]  Other examples include the usage of water absorption [ 26 ]  and thermal shape memory [ 27 ]  to demonstrate shape change. A 2015 study considered a 4D-printed capsule system that could release drugs on-demand at specific locations with a core-shell hydrogel. [ 28 ]  A 2014 study evaluated thermo-responsive poly(propylene fumarate) (PPF)-based system that released drugs in a controlled manner for treating the gastrointestinal tract. [ 29 ]


A  bong  (also known as a  water pipe ) is a filtration device generally used for smoking  cannabis ,  tobacco , or other herbal substances. [ 1 ]   In the bong shown in the photo, the smoke flows from the lower port on the left to the upper port on the right.


In construction and function, a bong is similar to a  hookah , except smaller and especially more portable. A bong may be constructed from any air- and water-tight vessel by adding a bowl and stem apparatus (or slide) [ 2 ]  which guides air downward to below water level whence it bubbles upward ("bubbler") during use. To get fresh air into the bong and harvest the last remaining smoke, a hole known as the "carburetor", "carb", "choke", "bink", "rush", "shotty", "kick hole", or simply "hole", somewhere on the lower part of the bong above water level, is first kept covered during the smoking process, then opened to allow the smoke to be drawn into the respiratory system. On bongs without such a hole, the bowl and/or the stem are removed to allow air from the hole that holds the stem.


Bongs have been in use by the  Hmong  in  Laos  and  Thailand , as well all over Africa, for centuries. [ 3 ]  One of the earliest recorded uses of the word in the West is in the McFarland Thai-English Dictionary, published in 1944, which describes one of the meanings of  bong  in the Thai language as, "a bamboo waterpipe for smoking kancha, tree, hashish, or the hemp-plant". A January 1971 issue of the  Marijuana Review  also used the term.


The word  bong  is an adaptation of the  Thai  word  bong  or  baung  ( Thai :  บ้อง ,  [bɔ̂ŋ] ), which refers to a cylindrical  wooden  tube,  pipe , or  container  cut from  bamboo , and which also refers to the bong used for smoking.


Excavations of a  kurgan  in Russia in 2013 revealed that  Scythian  tribal chiefs used gold vessels 2400 years ago to smoke cannabis and opium. The kurgan was discovered when construction workers were clearing land for the construction of a power line. [ 4 ]


During the reign of  Emperor Akbar , physician Hakim Abul Fath invented the waterpipe in  India , and discovered tobacco. Abul suggested that tobacco "smoke should be first passed through a small receptacle of water so that it would be rendered harmless". [ 5 ]  Other sources also show evidence of the invention of the waterpipe in  China  during the late  Ming dynasty  (16th century), along with tobacco, through Persia and the  Silk Road . [ citation needed ]  By the  Qing dynasty , it became the most popular method to smoke tobacco, but became less popular since the Republic era. While typically employed by commoners, the water pipe is known to have been preferred by  Empress Dowager Cixi  over snuff bottles or other methods of intake. According to the  Imperial Household Department , she was buried with at least three water pipes; some of her collections can be seen in the  Palace Museum .


The water pipe employed since the Qing dynasty can be divided into two types: the homemade bamboo bong commonly made and used by country people, and a more elegant metal version employed by Chinese merchants, urbanites, and nobility. Metal utensils are typically made out of bronze or brass, the nobility version of silver and decorated with jewels. Typically, the metal version is made out of the following components:


During a smoking session, the user may keep all equipment inside the rack and just hold the entire assembly (rack, pipe, and container) in one hand, lighting the bowl with a slow-burning paper wick (纸煤) lit over a coal stove.
Unlike in North America and the Southern Hemisphere, the water pipe is typically employed by older generations. [ citation needed ]


The water can trap some heavier particles and water-soluble molecules, preventing them from entering the smoker's airways. [ 6 ]  The mechanics of a bong are compared to those of a laboratory  gas washing  bottle. The user puts their mouth at the top and places the cannabis in the tube, as shown in the picture.


Bongs are often either  glass  or  plastic  that use a bowl, stem, and water to produce smoke. Most glass bongs are made from heat resistant  borosilicate glass , allowing the bong to withstand repeated use and heat exposure without breaking. After the bowl has been packed and water has been inserted into the bong, the substance is lit and the smoke is drawn through water to produce a smoother smoke than other methods of smoking do. [ 7 ]  To smoke a bong, the smoker must inhale in the bong so bubbles containing smoke begin to come from the stem. Once the bong has a fair amount of smoke built up, either the carb is uncovered or the stem is separated from the bong, allowing the remaining smoke to be inhaled.


However, a 2000  NORML - MAPS  cannabis study found that "water pipes filter out more psychoactive  THC  than they do other tars, thereby requiring users to smoke more to reach their desired effect". [ 8 ]  In the study, smoke from cannabis supplied by the  NIDA  was drawn through a number of smoking devices and analyzed. This study looked at the tar to cannabinoid ratio in the gas in output by various bongs, as well as unfiltered and filtered joints, and vaporizers. The results showed that only vaporizers produced a better tar to cannabinoid ratio than unfiltered joints, but that within the cannabinoids produced, even vaporizers warped the ratio of THC (the psychoactive component of the smoke) to  CBN  (capable of producing medical benefits but is not psychoactive) in favor of CBN. This showed an unfiltered joint had the best tar to THC ratio of all, and bongs were actually seriously detrimental in this respect.


MAPS [ 9 ]  also reviewed a study that examined the effects and composition of water-filtered and non-filtered  cannabis  and  tobacco smoke . It found that when  alveolar   macrophages  were exposed to unfiltered smoke, their ability to fight bacteria was reduced, unlike exposure to water-filtered smoke. It also found substantial epidemiological evidence of a lower incidence of  carcinoma  among tobacco smokers who used water-pipes, as opposed to  cigarettes ,  cigars , and regular  pipes . "It appears that water filtration can be effective in removing components from cannabis smoke that are known toxicants... The effectiveness of toxicant removal is related to the smoke's water contact area."


Specially designed water pipes, incorporating particulate filters and gas-dispersion  frits , would likely be most effective in this regard; the gas-dispersion frit serves to break up the smoke into very fine bubbles, thereby increasing its water-contact area. [ 9 ]  These frits are commonly referred to as  diffuser  for the way that they diffuse (or disperse) the smoke as it exits the downstem, and usually consist of small holes or slats at the end of the downstem. This study suggests that a bong's smoke is less harmful than unfiltered smoke.


Bongs that are cleaned regularly eliminates yeast, fungi, bacteria and pathogens that can cause several symptoms that vary from allergy to lung infection. [ 10 ]


It has been reported that it is possible to taste the chemicals in the water from a plastic water bottle left sitting in a hot car because "...chemicals have leached into the contents of the bottle." [ 11 ]  It was also reported that it is possible to imagine that this process of chemicals leaching into water occurring with "...a homemade water bottle bong where the bottle is routinely heated up." [ 11 ]


It was also reported that "According to  Cancer Research UK , plastic bottles heated up to 60°C contain unhealthy levels of toxic chemicals." [ 11 ]


In the  United States , under the Federal Drug Paraphernalia Statute, which is part of the  Controlled Substances Act , it is illegal to sell, transport through the mail, transport across state lines, import, or export drug paraphernalia. [ 12 ]


In countries where marijuana and hashish are illegal, some retailers specify that bongs are intended for use with tobacco in an attempt to circumvent laws against selling drug paraphernalia. While technically "bong" does not mean a device used for smoking mainly cannabis, drug-related connotations have been formed with the word itself (partly due to punning with Sanskrit  bhangah  "hemp"). Thus for fear of the law many  head shops  will not serve customers who use the word "bong" or "bongs", or any other word typically associated with illegal drug use. [ 13 ]


Some  brand name  bong manufacturers (notably RooR) have sought to curb the  counterfeit  market for their products by suing stores accused of selling fake merchandise. [ 14 ]


Breast milk-mediated drug delivery  refers to the use of  breast milk  to transport a pharmaceutical compound, protein, or other treatment to achieve a desired effect. Delivery of these substances via milk provides an oral alternative for transport of a compound to the gut, specifically in infants. Breast milk-mediated drug delivery provides a way for pharmaceuticals and proteins to travel through the  gastrointestinal system  of an infant while minimizing the potential for irritation within gastrointestinal tissue.


There are multiple routes that a treatment can enter into the breast milk. One route is through a  breastfeeding  mother. When a mother that is  lactating  receives a treatment including but not limited to  prescription medication ,  over-the-counter medications , or  herbal remedies , these compounds can make their way into the mother’s milk and are subsequently delivered to an infant via breastfeeding. [ 1 ]  The amount of treatment transferred into the milk depends on the substance, but it is noted that these compounds can have an effect on the infant consuming the breast milk. [ 1 ]


A common method for intended addition of treatment into breast milk involves the use of donor breast milk in combination with the compound of interest for treatment. Addition of  antibiotics ,  oligosaccharides ,  cytokines , or selected proteins into donor breast milk for consumption can have a variety of effects on the patient consuming it, typically an infant. [ 2 ] [ 3 ] [ 4 ]  This method has also been studied for use in adults, specifically for the delivery of poorly water-soluble drugs. [ 5 ] [ 6 ]  While this method allows for the highest level of modularity among breast milk treatments, the effects of these treatments on various factors such as the  microbiome  of the milk base must be acknowledged to limit potential side effects.


Other novel technologies for drug delivery via breast milk involve the use of external objects to mediate the addition of compounds into the milk being consumed. One of these technologies is a  nipple shield . A nipple shield is placed onto the nipple of a breastfeeding mother, releasing treatment into the milk as the mother breastfeeds an infant. [ 7 ]  Many of these novel technologies still require testing for efficacy and safety before any potential commercial application.


Breast milk fortification refers to the addition of nutrients such as proteins, oligosaccharides, or cytokines into breast milk to supplement the natural contents of the milk. [ 2 ] [ 3 ] [ 4 ]  This practice is common in babies that have been born prematurely, with a major goal of this practice being growth promotion. [ 8 ]  These infants do not receive enough nutrients for proper growth and development from a normal breastfeeding regimen, potentially leading to neural and/or cognitive impairment. [ 8 ] [ 9 ]  Thus, supplementation of breast milk with proteins, calcium, phosphate, and other nutrients can help to remedy this issue. [ 8 ] [ 9 ]


Studies have shown that human milk fortification can improve the short-term growth of infants, though future research is needed to determine long term growth effects. [ 10 ]  There is variation with results from case to case, however. The nonstandard concentration of proteins across different mothers’ breast milk can lead to issues when pinpointing the amount of nutrients an infant is receiving. [ 8 ]  While there is a standard fortification level used in most cases, “Adjustable Fortification and “Targeted Fortification” are being researched as ways to provide targeted individual fortification to suit the needs of each individual being treated. [ 8 ]


One particular compound that can be used to fortify breast milk is  Human Milk Oligosaccharide . These prebiotics are found naturally in human breast milk, and provide a variety of health benefits to newborns, including modulation of the immune system, fighting  viral infections , and promoting epithelial cell maturation. [ 3 ]  These prebiotics, which have been confirmed to be safe and tolerable for infants, are a leading candidate for breast milk fortification.


Antibiotics can be added into breast milk as a preventative measure for the development of infections such as Subsequent Late Onset Sepsis and Necrotizing Enterocolitis. [ 11 ]  Antibiotic treatment is a very common treatment for premature babies, though dosing regimen and treatment length tend to be “arbitrary”. [ 11 ]  While potentially detrimental to the health of the gut microbiome if given in excess, the administration of antibiotics within breast milk can help to prevent infection in newborns. [ 12 ]


Antibiotics are used as the most common treatment for prematurely born infants in  Neonatal Intensive Care Units  – used as a preventative measure for infection. These treatments usually contain empirical antibiotics and are often given to infants that do not show signs of  bacterial infection . [ 13 ]  Studies are currently being performed to determine the optimal length of treatment for infants to prevent infection while maintaining gut health, specifically in the microbiome. [ 11 ] [ 12 ] [ 13 ]


Stability of milk-drug formulations is another factor that contributes to the effectiveness of antibiotic addition into breast milk. Many orally delivered antibiotics show poor solubility in aqueous environments, leading to increased research into lipid-based drug delivery systems. [ 6 ]  Milk formulations, which contain varying levels of natural lipids, have been shown to increase delivery efficacy of antibiotic compounds, while maintaining stability and integrity of the colloidal structure that makes up milk. [ 6 ]


Addition of carefully selected compounds into breast milk have been shown to not only stimulate growth within an infant, but also help to prevent infections from forming, specifically in the gut. Babies born very prematurely are at high risk for these infections, particularly  necrotizing enterocolitis . [ 14 ]  Necrotizing enterocolitis is an intestinal condition that develops in some premature babies, and is characterized by  inflammation , bacterial infection, potential intestinal injury, and in extreme cases, death. [ 13 ]


The development of necrotizing enterocolitis has also been linked to the microbiome of the gut in the affected population. [ 11 ] [ 12 ]  High levels of antibiotic administration have been shown to reduce the variety of bacteria present within the gut microbiome, and this lower level of diversity found in these infants can increase their susceptibility to necrotizing enterocolitis. [ 11 ]  Studies have indicated that time of administration of antibiotics as well as type of antibiotic can impact the incidence of the condition. [ 12 ]


Prevention of necrotizing enterocolitis in premature infants is mainly achieved through the addition of predetermined compounds into donor breast milk that is fed to premature infants at risk for the condition. Compounds such as  immunoglobulins  have been shown to reduce Necrotizing Enterocolitis incidence, as well as severity in those that still develop the condition. [ 2 ]  Studies have shown that the addition of  IgA  and  IgG  into a premature infant’s feed can lower the incidence of the disease in the population of infants that are at the highest risk for the condition (very premature, low birth weight). [ 2 ]


One of the main issues surrounding breast milk-mediated drug delivery is the uncertainty of the composition of the milk acting as the vessel for the treatment. Since every breastfeeding mother experiences different external conditions such as diet and environment, as well as differences in genetics, breast milk from every mother will have a different composition, making it difficult to create replicable treatments from infant to infant. [ 8 ]  Donor breast milk encounters this same issue, with infant formula acting as a potential avenue for stabilization of treatment levels.


Improper administration of antibiotics into an infant’s gastrointestinal system can increase the susceptibility of infants to gut infections. This is due to the lowered diversity and viability of organisms found within the gut, also known as the microbiome. [ 11 ] [ 12 ]  Especially prevalent in  preterm infants , this deviation from a healthy microbiome can lead to adverse effects during the beginning of an infant’s life. These infants may be predisposed to conditions such as Necrotizing Enterocolitis due to higher growth of pathogenic bacteria within the gut, as well as a lower count of commensal bacteria growth. [ 11 ]


New breast milk-mediated therapies are still in development, aiming to take advantage of properties that are unique to this form of treatment. One such technology is the use of  exosomes  found in milk to transport molecules such as  mRNA ,  DNA , and proteins to an area of interest. [ 15 ]  This research proposes the use of milk, as a scalable source of exosomes for a method of targeted drug delivery with higher efficacy than standard oral methods. [ 15 ]  These novel therapies may provide a way to achieve a low-cost method to deliver drugs with poor water solubility into the body, with potential use as a low-cost alternative. [ 5 ]


Cell encapsulation  is a possible solution to  graft rejection  in tissue engineering applications.  Cell  microencapsulation  technology involves  immobilization  of cells within a polymeric  semi-permeable membrane . It permits the bidirectional  diffusion  of molecules such as the influx of oxygen, nutrients,  growth factors  etc. essential for cell  metabolism  and the outward diffusion of waste products and  therapeutic  proteins.  At the same time, the semi-permeable nature of the membrane prevents immune cells and  antibodies  from destroying the encapsulated cells, regarding them as foreign invaders.


Cell encapsulation could reduce the need for long-term use of  immunosuppressive drugs  after an  organ transplant  to control side effects.


In 1933 Vincenzo Bisceglie made the first attempt to encapsulate cells in polymer membranes. He demonstrated that  tumor  cells in a polymer structure transplanted into pig  abdominal  cavity remained viable for a long period without being rejected by the  immune system . [ 1 ]


Thirty years later in 1964, the idea of encapsulating cells within ultra thin polymer membrane microcapsules so as to provide immunoprotection to the cells was then proposed by  Thomas Chang  who introduced the term " artificial cells " to define this concept of bioencapsulation. [ 2 ]  He suggested that these artificial cells produced by a drop method not only protected the encapsulated cells from immunorejection but also provided a high surface-to-volume relationship enabling good mass transfer of oxygen and nutrients. [ 2 ]
Twenty years later, this approach was successfully put into practice in small animal models when alginate-polylysine-alginate (APA) microcapsules immobilizing xenograft  islet  cells were developed. [ 3 ]  The study demonstrated that when these microencapsulated islets were implanted into  diabetic  rats, the cells remained viable and controlled  glucose  levels for several weeks.
Human trials utilising encapsulated cells were performed in 1998. [ 4 ] [ 5 ] [ 6 ]  Encapsulated cells expressing a cytochrome P450 enzyme to locally activate an anti-tumour prodrug were used in a trial for advanced, non-resectable pancreatic cancer. Approximately a doubling of survival time compared to historic controls was demonstrated.


Questions could arise as to why the technique of encapsulation of cells is even required when therapeutic products could just be injected at the site. An important reason for this is that the encapsulated cells would provide a source of sustained continuous release of therapeutic products for longer durations at the site of implantation. Another advantage of cell microencapsulation technology is that it allows the loading of non-human and genetically modified cells into the polymer matrix when the availability of donor cells is limited. [ 7 ]  Microencapsulation is a valuable technique for local, regional and oral delivery of therapeutic products as it can be implanted into numerous tissue types and organs. For prolonged  drug delivery  to the treatment site, implantation of these drug loaded  artificial cells would be more cost effective in comparison to direct drug delivery. Moreover, the prospect of implanting artificial cells with similar chemical composition in several patients irrespective of their leukocyte antigen could again allow reduction in costs. [ 7 ]


The potential of using cell microencapsulation in successful clinical applications can be realized only if several requirements encountered during the development process are optimized such as the use of an appropriate  biocompatible  polymer to form the mechanically and chemically stable semi-permeable matrix, production of uniformly sized microcapsules, use of an appropriate immune-compatible polycations cross-linked to the encapsulation polymer to stabilized the capsules, selection of a suitable cell type depending on the situation.


The use of the best biomaterial depending on the application is crucial in the development of drug delivery systems and tissue engineering. The polymer  alginate  is very commonly used due to its early discovery, easy availability and low cost but other materials such as cellulose sulphate,  collagen ,  chitosan ,  gelatin  and  agarose  have also been employed.


Several groups have extensively studied several natural and synthetic polymers with the goal of developing the most suitable biomaterial for cell microencapsulation. [ 8 ] [ 9 ]  Extensive work has been done using alginates which are regarded as the most suitable biomaterials for cell microencapsulation due to their abundance, excellent biocompatibility and   biodegradability  properties. Alginate is a natural polymer which can be extracted from seaweed and  bacteria [ 10 ]  with numerous compositions based on the isolation source. [ 10 ]


Alginate is not free from all criticism. Some researchers believe that alginates with high-M content could produce an  inflammatory  response [ 11 ] [ 12 ]  and an abnormal cell growth [ 13 ]  while some have demonstrated that  alginate  with high-G content lead to an even higher cell overgrowth [ 14 ] [ 15 ]  and inflammatory reaction in vivo as compared to intermediate-G alginates. [ 16 ] [ 17 ]
Even ultrapure alginates may contain  endotoxins , and  polyphenols  which could compromise the biocompatibility of the resultant cell microcapsules. [ 15 ] [ 18 ] [ 19 ]  It has been shown that even though purification processes successfully lower endotoxin and polyphenol content in the processed alginate, it is difficult to lower the protein content [ 18 ]  and the purification processes could in turn modify the properties of the biomaterial. [ 19 ]  Thus it is essential that an effective purification process is designed so as to remove all the contaminants from alginate before it can be successfully used in clinical applications.


Researchers have also been able to develop alginate microcapsules with an altered form of alginate with enhanced biocompatibility and higher resistance to osmotic swelling. [ 20 ] [ 21 ]
Another approach to increasing the biocompatibility of the membrane biomaterial is through surface modification of the capsules using  peptide  and protein molecules which in turn controls the proliferation and rate of differentiation of the encapsulated cells. One group that has been working extensively on coupling the amino acid sequence Arg-Gly-Asp (RGD) to alginate hydrogels demonstrated that the cell behavior can be controlled by the RGD density coupled on the alginate gels. Alginate microparticles loaded with myoblast cells and functionalized with RGD allowed control over the growth and differentiation of the loaded cells. [ 22 ] [ 23 ]
Another vital factor that controls the use of cell microcapsules in clinical applications is the development of a suitable immune-compatible polycation to coat the otherwise highly porous alginate beads and thus impart stability and immune protection to the system. [ 24 ]   Poly-L-lysine  is the most commonly used polycation but its low biocompatibility restricts the successful clinical use of these PLL formulated microcapsules which attract inflammatory cells thus inducing necrosis of the loaded cells. [ 25 ]  Studies have also shown that alginate-PLL-alginate (APA) microcapsules demonstrate low mechanical stability and short term durability. Thus several research groups have been looking for alternatives to PLL and have demonstrated promising results with poly-L-ornithine [ 26 ]  and poly(methylene-co-guanidine) hydrochloride [ 27 ]  by fabricating durable microcapsules with high  and controlled mechanical strength for cell encapsulation.


Several groups have also investigated the use of  chitosan  which is a naturally derived polycation as a potential replacement for PLL to fabricate alginate-chitosan (AC) microcapsules for cell delivery applications. [ 28 ] [ 29 ]  However, studies have also shown that the stability of this AC membrane is again limited [ 30 ] [ 31 ]  and one group demonstrated that modification of this alginate-chitosan microcapsules with  genipin , a naturally occurring iridoid glucosid from gardenia fruits, to form genipin cross-linked alginate-chitosan (GCAC) microcapsules could augment stability of the cell loaded microcapsules. [ 30 ]


Collagen, a major protein component of the ECM, provides support to tissues like skin, cartilage, bones, blood vessels and ligaments and is thus considered a model scaffold or matrix for tissue engineering due to its properties of biocompatibility,  biodegradability  and ability to promote cell binding. [ 32 ]  This ability allows chitosan to control distribution of cells inside the polymeric system. Thus, Type-I collagen obtained from animal tissues is now successfully being used commercially as tissue engineered biomaterial for multiple applications. [ 33 ]  Collagen has also been used in nerve repair [ 34 ]  and bladder engineering. [ 27 ]   Immunogenicity  has limited the applications of collagen. Gelatin has been considered as an alternative for that reason. [ 35 ]


Gelatin  is prepared from the denaturation of  collagen  and many desirable properties such as  biodegradability , biocompatibility, non-immunogenity in physiological
environments, and easy processability make this polymer a good choice for tissue engineering applications. [ 36 ]  It is used in engineering tissues for the skin, bone and cartilage and is used commercially for skin replacements. [ 37 ]


Chitosan  is a polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is derived from the N-deacetylation of  chitin  and  has been used for several applications such as  drug delivery , [ 38 ]  space-filling implants [ 39 ]  and in wound dressings. [ 40 ]  However, one drawback of this polymer is its weak mechanical properties  and is thus often combined with other polymers  such  collagen  to form a polymer with stronger mechanical properties for cell encapsulation applications. [ 41 ]


Agarose  is a  polysaccharide   derived from seaweed used for nanoencapsulation of cells and the cell/agarose suspension [ 42 ]   can be modified to form  microbeads by reducing the temperature during preparation. [ 43 ]  However, one drawback with the microbeads so obtained is the possibility of cellular protrusion through the  polymeric  matrix wall after formation of the capsules.


Cellulose sulphate is derived from cotton and, once processed appropriately, can be used as a biocompatible base in which to suspend cells. When the poly-anionic cellulose sulphate solution is immersed in a second, poly-cationic solution (e.g. pDADMAC), a semi-permeable membrane is formed around the suspended cells as a result of gelation between the two poly-ions. Both mammalian cell lines and bacterial cells remain viable and continue to replicate within the capsule membrane in order to fill-out the capsule. As such, in contrast to some other encapsulation materials, the capsules can be used to grow cells and act as such like a mini-bioreactor. The biocompatible nature of the material has been demonstrated by observation during studies using the cell-filled capsules themselves for implantation as well as isolated capsule material. [ 44 ]  Capsules formed from cellulose sulphate have been successfully used, showing safety and efficacy, in clinical and pre-clinical trials in both humans and animals, primarily as anti-cancer treatments, but also exploring possible uses for gene therapy or antibody therapies. [ 4 ] [ 45 ] [ 46 ] [ 47 ] [ 48 ]  Using cellulose sulphate it has been possible to manufacture encapsulated cells as a pharmaceutical product at large scale and fulfilling Good Manufacturing Process (cGMP) standards. This was achieved by the company  Austrianova  in 2007. [ 49 ]


The use of an ideal high quality biomaterial with the inherent properties of biocompatibility is the most crucial factor that governs the long term efficiency of this technology. An ideal biomaterial for cell encapsulation should be one that is totally  biocompatible , does not trigger an immune response in the host and does not interfere with cell  homeostasis  so as to ensure high cell viability. [ 50 ]  However, one major limitation has been the inability to reproduce the different biomaterials and the requirements to obtain a better understanding of the chemistry and biofunctionality of the biomaterials and the  microencapsulation  system. [ 42 ]   Several studies demonstrate that surface modification of these cell containing microparticles allows control over the growth and cellular differentiation. [ 42 ] [ 51 ]  of the encapsulated cells. [ 52 ]


One study proposed the use of  zeta potential  which measures the  electric charge  of the microcapsule as a means to predict the interfacial reaction between microcapsule and the surrounding tissue and in turn the biocompatibility of the delivery system. [ 53 ]


A fundamental criterion that must be established while developing any device with a  semi-permeable membrane  is to adjust the permeability of the device in terms of entry and exit of molecules. [ 54 ] [ 55 ]  It is essential that the cell microcapsule is designed with uniform thickness and should have a control over both the rate of molecules entering the capsule necessary for cell viability and the rate of  therapeutic  products and waste material exiting the capsule membrane. Immunoprotection of the loaded cell is the key issue that must be kept in mind while working on the permeability of the encapsulation membrane as not only immune cells but also  antibodies  and  cytokines  should be prevented entry into the microcapsule which in fact depends on the pore size of the biomembrane. [ 55 ]


It has been shown that since different cell types have different metabolic requirements, thus depending on the cell type encapsulated in the membrane the permeability of the membrane has to be optimized. [ 56 ]  Several groups have been dedicated towards the study of membrane permeability of cell microcapsules [ 51 ] [ 52 ] [ 57 ]  and although the role of permeability of certain essential elements like oxygen has been demonstrated, [ 58 ]  the permeability requirements of each cell type are yet to be determined.


Sodium Citrate is used for degradation of alginate beads after encapsulation of cells. [ 59 ]  In order to determine viability of the cells or for further experimentation. Concentrations of approximately 25mM are used to dissolve the alginate spheres and the solution is spun down using a centrifuge so the sodium citrate can be removed and the cells can be collected.


It is essential that the microcapsules have adequate membrane strength (mechanical stability) to endure physical and  osmotic stress  such as during the exchange of nutrients and waste products. The microcapsules should be strong enough and should not rupture on implantation as this could lead to an immune rejection of the encapsulated cells. [ 55 ]  For instance, in the case of  xenotransplantation , a tighter more stable membrane would be required in comparison to  allotransplantation . Also, while investigating the potential of using APA microcapsules loaded with bile salt hydrolase (BSH) overproducing active  Lactobacillus  plantarum 80 cells, in a simulated  gastro intestinal tract  model for oral delivery applications, the mechanical integrity and shape of the microcapsules was evaluated. It was shown that APA microcapsules could potentially be used in the oral delivery of live bacterial cells. [ 60 ]  However, further research proved that the GCAC microcapsules possess a higher mechanical stability as compared to APA microcapsules for oral delivery applications. [ 61 ]  Martoni et al. were experimenting with bacteria-filled capsules that would be taken by mouth to reduce serum cholesterol.
The capsules were pumped through a series of vessels simulating the human GI tract to determine how well the capsules would survive in the body. Extensive research into the mechanical properties of the biomaterial to be used for cell microencapsulation is necessary to determine the durability of the microcapsules during production and especially for in vivo applications where a sustained release of the therapeutic product over long durations is required.
van der Wijngaart et al. [ 57 ]  grafted a solid, but permeable, shell around the cells to provide increased mechanical strength.


Droplet-based microfluidics can be used to generate microparticles with repeatable size. [ 57 ]


Eletrospraying is used to create alginate spheres by pumping an alginate solution through a needle. A source of high voltage usually provided by a clamp attached to the needle is used to generate an electric potential with the alginate falling from the needle tip into a solution that contains a ground.  Calcium chloride is used as cross linking solution in which the generated capsules drop into where they harden after approximately 30 minutes. Beads are formed from the needle due to charge and surface tension. [ 62 ]


The diameter of the microcapsules is an important factor that influences both the immune response towards the cell microcapsules as well as the mass transport across the capsule membrane. Studies show that the cellular response to smaller capsules is much lesser as compared to larger capsules [ 63 ]  and in general the diameter of the cell loaded microcapsules should be between 350-450 μm so as to enable effective diffusion across the semi-permeable membrane. [ 64 ] [ 65 ]


The cell type chosen for this technique depends on the desired application of the cell microcapsules. The cells put into the capsules can be from the patient ( autologous  cells), from another donor (allogeneic cells) or from other species (xenogeneic cells). [ 66 ]  The use of autologous cells in microencapsulation therapy is limited by the availability of these cells and even though xenogeneic cells are easily accessible, danger of possible transmission of  viruses , especially  porcine  endogenous  retrovirus  to the patient restricts their clinical application, [ 67 ]  and after much debate several groups have concluded that studies should involve the use of allogeneic instead of xenogeneic cells. [ 68 ]  Depending on the application, the cells can be genetically altered to express any required protein. [ 69 ]  However, enough research has to be carried out to validate the safety and stability of the expressed gene before these types of cells can be used.


This technology has not received approval for clinical trial because of the high immunogenicity of cells loaded in the capsules. They secrete  cytokines  and produce a severe  inflammatory  reaction at the implantation site around the capsules, in turn leading to a decrease in viability of the encapsulated cells. [ 15 ] [ 70 ]  One promising approach being studied is the administration of anti-inflammatory drugs to reduce the immune response produced due to administration of the cell loaded microcapsules. [ 71 ] [ 72 ]  Another approach which is now the focus of extensive research is the use of  stem cells  such as mesenchymal stem cells for long term cell microencapsulation and cell therapy applications in hopes of reducing the immune response in the patient after implantation. [ 73 ]  Another issue which compromises long term viability of the microencapsulated cells is the use of fast proliferating cell lines which eventually fill up the entire system and lead to decrease in the  diffusion  efficiency across the semi-permeable membrane of the capsule. [ 69 ]  A solution to this could be in the use of cell types such as  myoblasts  which do not proliferate after the microencapsulation procedure.


Probiotics  are increasingly being used in numerous dairy products such as ice cream, milk powders, yoghurts, frozen dairy desserts and cheese due to their important health benefits. But, low viability of probiotic  bacteria  in the food still remains a major hurdle. The  pH , dissolved oxygen content, titratable acidity, storage temperature, species and strains of associative fermented dairy product organisms and concentration of lactic and acetic acids are some of the factors that greatly affect the probiotic viability in the product. [ 74 ] [ 75 ] [ 76 ]  As set by  Food and Agriculture Organization  (FAO) of the United Nations and the  World Health Organization  (WHO), the standard in order to be considered a health food with probiotic addition, the product should contain per gram at least 10 6 -10 7   cfu  of  viable  probiotic bacteria. [ 77 ]  It is necessary that the bacterial cells remain stable and healthy in the manufactured product, are sufficiently  viable  while moving through the upper digestive tract and are able to provide positive effects upon reaching the intestine of the host. [ 78 ]


Cell microencapsulation technology has successfully been applied in the food industry for the encapsulation of live probiotic bacteria cells to increase viability of the bacteria during processing of dairy products and for targeted delivery to the gastrointestinal tract. [ 79 ]


Apart from dairy products, microencapsulated probiotics have also been used in non-dairy products, such as  TheresweetTM  which is a  sweetener . It can be used as a convenient vehicle for delivery of encapsulated  Lactobacillus  to the intestine although it is not itself a dairy product.


The potential of using  bioartificial pancreas , for treatment of  diabetes mellitus , based on encapsulating  islet  cells within a semi permeable membrane is extensively being studied by scientists. These devices could eliminate the need for of  immunosuppressive drugs  in addition to finally solving the problem of shortage of organ donors. The use of microencapsulation would protect the islet cells from immune rejection as well as allow the use of animal cells or genetically modified insulin-producing cells. [ 80 ]  It is hoped that development of these islet encapsulated microcapsules could prevent the need for the insulin injections needed several times a day by type 1 diabetic patients. [ 66 ]  The  Edmonton protocol  involves implantation of human islets extracted from cadaveric donors and has shown improvements towards the treatment of type 1 diabetics who are prone to  hypoglycemic  unawareness. [ 81 ]  However, the two major hurdles faced in this technique are the limited availability of donor organs and with the need for  immunosuppresents  to prevent an immune response in the patient's body.