Pericytes (formerly called Rouget cells) are multi-functional mural cells of the microcirculation that wrap around the endothelial cells that line the capillaries throughout the body. Pericytes are embedded in the basement membrane of blood capillaries, where they communicate with endothelial cells by means of both direct physical contact and paracrine signaling. The morphology, distribution, density and molecular fingerprints of pericytes vary between organs and vascular beds. Pericytes help to maintain homeostatic and hemostatic functions in the brain, one of the organs with higher pericyte coverage, and also sustain the blood–brain barrier. These cells are also a key component of the neurovascular unit, which includes endothelial cells, astrocytes, and neurons. Pericytes have been postulated to regulate capillary blood flow and the clearance and phagocytosis of cellular debris in vitro.
Pericytes stabilize and monitor the maturation of endothelial cells by
means of direct communication between the cell membrane as well as
through paracrine signaling. A deficiency of pericytes in the central nervous system can cause increased permeability of the blood–brain barrier.
Structure
Gap cell junction created between two neighboring cells by connexin.
In the central nervous system
(CNS), pericytes wrap around the endothelial cells that line the inside
of the capillary. These two types of cells can be easily distinguished
from one another based on the presence of the prominent round nucleus of the pericyte compared to the flat elongated nucleus of the endothelial cells.
Pericytes also project finger-like extensions that wrap around the
capillary wall, allowing the cells to regulate capillary blood flow.
Both pericytes and endothelial cells share a basement membrane
where a variety of intercellular connections are made. Many types of integrin molecules facilitate communication between pericytes and endothelial cells separated by the basement membrane.
Pericytes can also form direct connections with neighboring cells by
forming peg and socket arrangements in which parts of the cells
interlock, similar to the gears of a clock. At these interlocking
sites, gap junctions can be formed, which allow the pericytes and neighboring cells to exchange ions and other small molecules. Important molecules in these intercellular connections include N-cadherin, fibronectin, connexin and various integrins.
In some regions of the basement membrane, adhesion plaques composed of fibronectin can be found. These plaques facilitate the connection of the basement membrane to the cytoskeletal structure composed of actin, and the plasma membrane of the pericytes and endothelial cells.
Function
Skeletal muscle regeneration and fat formation
Pericytes in the skeletal striated muscle are of two distinct populations, each with its own role. The first pericyte subtype (Type-1) can differentiate into fat cells while the other (Type-2) into muscle cells. Type-1 characterized by negative expression for nestin
(PDGFRβ+CD146+Nes-) and type-2 characterized by positive expression for
nestin (PDGFRβ+CD146+Nes+). While both types are able to proliferate in
response to glycerol or BaCl2-induced
injury, type-1 pericytes give rise to adipogenic cells only in response
to glycerol injection and type-2 become myogenic in response to both
types of injury. The extent to which type-1 pericytes participate in fat accumulation is not known.
Angiogenesis and the survival of endothelial cells
Pericytes are also associated with endothelial cell differentiation and multiplication, angiogenesis, survival of apoptotic
signals and travel. Certain pericytes, known as microvascular
pericytes, develop around the walls of capillaries and help to serve
this function. Microvascular pericytes may not be contractile cells, as
they lack alpha-actin isoforms, structures that are common amongst other contractile cells. These cells communicate with endothelial cells via gap junctions, and in turn cause endothelial cells to proliferate or be selectively inhibited. If this process did not occur, hyperplasia and abnormal vascular morphogenesis could result. These types of pericyte can also phagocytose exogenous proteins. This suggests that the cell type might have been derived from microglia.
A lineage relationship to other cell types has been proposed, including smooth muscle cells, neural cells, NG2 glia, muscle fibers, adipocytes, as well as fibroblasts and other mesenchymal stem cells.
However, whether these cells differentiate into each other is an
outstanding question in the field. Pericytes' regenerative capacity is
affected by aging.
Such versatility is useful, as they actively remodel blood vessels
throughout the body and can thereby blend homogeneously with the local tissue environment.
Aside from creating and remodeling blood vessels, pericytes have
been found to protect endothelial cells from death via apoptosis or cytotoxic elements. It has been shown in vivo that pericytes release a hormone known as pericytic aminopeptidase N/pAPN that may help to promote angiogenesis. When this hormone was mixed with cerebral
endothelial cells as well as astrocytes, the pericytes grouped into
structures that resembled capillaries. Furthermore, when the
experimental group contained all of the following with the exception of
pericytes, the endothelial cells would undergo apoptosis.
It was thus concluded that pericytes must be present to ensure the
proper function of endothelial cells, and astrocytes must be present to
ensure that both remain in contact. If not, then proper angiogenesis
cannot occur. It has also been found that pericytes contribute to the survival of endothelial cells, as they secrete the protein Bcl-w during cellular crosstalk. Bcl-w is an instrumental protein in the pathway that enforces VEGF-A expression and discourages apoptosis. Although there is some speculation as to why VEGF is directly responsible for preventing apoptosis, it is believed to be responsible for modulating apoptotic signal transduction pathways and inhibiting activation of apoptosis-inducing enzymes. Two biochemical mechanisms utilized by VEGF to accomplish this would be phosphorylation of extracellular regulatory kinase 1
(ERK-1, also known as MAPK3), which sustains cell survival over time,
and inhibition of stress-activated protein kinase/c-jun-NH2 kinase,
which also promotes apoptosis.
Blood–brain barrier
Pericytes play a crucial role in the formation and functionality of the blood–brain barrier.
This barrier is composed of endothelial cells and ensures the
protection and functionality of the brain and central nervous system. It
has been found that pericytes are crucial to the postnatal formation of
this barrier. Pericytes are responsible for tight junction formation and vesicle trafficking amongst endothelial cells. Furthermore, they allow the formation of the blood–brain barrier by inhibiting the effects of CNS immune cells
(which can damage the formation of the barrier) and by reducing the
expression of molecules that increase vascular permeability.
Aside from blood–brain barrier formation, pericytes also play an
active role in its functionality. Animal models of developmental loss of
pericytes show increased endothelial transcytosis, as well as skewed
arterio-venous zonation, increased expression of leukocyte adhesion
molecules and microaneurysms.Loss or dysfunction of pericytes is also theorized to contribute to neurodegenerative diseases such as Alzheimer's, Parkinson's and ALS through breakdown of the blood-brain barrier.
Blood flow
Increasing
evidence suggests that pericytes can regulate blood flow at the
capillary level. For the retina, movies have been published
showing that pericytes constrict capillaries when their membrane
potential is altered to cause calcium influx, and in the brain it has
been reported that neuronal activity increases local blood flow by
inducing pericytes to dilate capillaries before upstream arteriole
dilation occurs.
This area is controversial, with a 2015 study claiming that pericytes
do not express contractile proteins and are not capable of contraction
in vivo,
although the latter paper has been criticised for using a highly
unconventional definition of pericyte which explicitly excludes
contractile pericytes.
It appears that different signaling pathways regulate the constriction
of capillaries by pericytes and of arterioles by smooth muscle cells.
Recent studies on rats have found such a signaling pathway in which
after spinal cord injury and induced hypoxia below the injury, there is
excess activity of monoamine receptors on pericytes which locally
constricts capillaries and reduces blood flow to ischemic levels.
Pericytes are important in maintaining circulation. In a study
involving adult pericyte-deficient mice, cerebral blood flow was
diminished with concurrent vascular regression due to loss of both
endothelia and pericytes. Significantly greater hypoxia was reported in
the hippocampus of pericyte-deficient mice as well as inflammation, and learning and memory impairment.
Clinical significance
Because
of their crucial role in maintaining and regulating endothelial cell
structure and blood flow, abnormalities in pericyte function are seen in
many pathologies. They may either be present in excess, leading to
diseases such as hypertension and tumor formation, or in deficiency,
leading to neurodegenerative diseases.
Hemangioma
The clinical phases of hemangioma
have physiological differences, correlated with immunophenotypic
profiles by Takahashi et al. During the early proliferative phase (0–12
months) the tumors express proliferating cell nuclear antigen
(pericytesna), vascular endothelial growth factor (VEGF), and type IV
collagenase, the former two localized to both endothelium and pericytes,
and the last to endothelium. The vascular markers CD31, von Willebrand
factor (vWF), and smooth muscle actin (pericyte marker) are present
during the proliferating and involuting phases, but are lost after the
lesion is fully involuted.
Hemangiopericytoma
Image
of a solitary fibrous tumour that is most likely a hemangiopericytoma.
It surrounds a staghorn-shaped blood vessel, which results from the
arrangement of pericytes around the vessel
Hemangiopericytoma
is a rare vascular neoplasm, or abnormal growth, that may either be
benign or malignant. In its malignant form, metastasis to the lungs,
liver, brain, and extremities may occur. It most commonly manifests
itself in the femur and proximal tibia as a bone sarcoma, and is usually
found in older individuals, though cases have been found in children.
Hemangiopericytoma is caused by the excessive layering of sheets of
pericytes around improperly formed blood vessels. Diagnosis of this
tumor is difficult because of the inability to distinguish pericytes
from other types of cells using light microscopy. Treatment may involve
surgical removal and radiation therapy, depending on the level of bone
penetration and stage in the tumor's development.
Diabetic retinopathy
The
retina of diabetic individuals often exhibits loss of pericytes, and
this loss is a characteristic factor of the early stages of diabetic retinopathy.
Studies have found that pericytes are essential in diabetic individuals
to protect the endothelial cells of retinal capillaries. With the loss
of pericytes, microaneurysms form in the capillaries. In response, the
retina either increases its vascular permeability, leading to swelling
of the eye through a macular edema, or forms new vessels that permeate into the vitreous membrane of the eye. The end result is reduction or loss of vision. While it is unclear why pericytes are lost in diabetic patients, one hypothesis is that toxic sorbitol and advanced glycation end-products (AGE) accumulate in the pericytes. Because of the build-up of glucose, the polyol pathway
increases its flux, and intracellular sorbitol and fructose accumulate.
This leads to osmotic imbalance, which results in cellular damage. The
presence of high glucose levels also leads to the buildup of AGE's,
which also damage cells.
Neurodegenerative diseases
Studies
have found that pericyte loss in the adult and aging brain leads to the
disruption of proper cerebral perfusion and maintenance of the
blood–brain barrier, which causes neurodegeneration and
neuroinflammation.
The apoptosis of pericytes in the aging brain may be the result of a
failure in communication between growth factors and receptors on
pericytes. Platelet-derived growth factor B (PDGFB)
is released from endothelial cells in brain vasculature and binds to
the receptor PDGFRB on pericytes, initiating their proliferation and
investment in the vasculature.
Immunohistochemical studies of human tissue from Alzheimer's
disease and amyotrophic lateral sclerosis show pericyte loss and
breakdown of the blood-brain barrier. Pericyte-deficient mouse models
(which lack genes encoding steps in the PDGFB:PDGFRB signalling cascade)
and have an Alzheimer's-causing mutation have exacerbated
Alzheimer's-like pathology compared to mice with normal pericyte
coverage and an Alzheimer's-causing mutation.
Stroke
In conditions of stroke,
pericytes constrict brain capillaries and then die, which may lead to a
long-lasting decrease of blood flow and loss of blood–brain barrier
function, increasing the death of nerve cells.
Research
Endothelial and pericyte interactions
Endothelial
cells and pericytes are interdependent and failure of proper
communication between the two cell types can lead to numerous human
pathologies.
There are several pathways of communication between the endothelial cells and pericytes. The first is transforming growth factor (TGF) signaling, which is mediated by endothelial cells. This is important for pericyte differentiation. Angiopoietin 1 and Tie-2 signaling is essential for maturation and stabilization of endothelial cells. Platelet-derived growth factor
(PDGF) pathway signaling from endothelial cells recruits pericytes, so
that pericytes can migrate to developing blood vessels. If this pathway
is blocked, it leads to pericyte deficiency. Sphingosine-1-phosphate (S1P) signaling also aids in pericyte recruitment by communication through G protein-coupled receptors. S1P sends signals through GTPases that promote N-cadherin trafficking to endothelial membranes. This trafficking strengthens endothelial contacts with pericytes.
Communication between endothelial cells and pericytes is vital.
Inhibiting the PDGF pathway leads to pericyte deficiency. This causes
endothelial hyperplasia, abnormal junctions, and diabetic retinopathy. A lack of pericytes also causes an upregulation of vascular endothelial growth factor (VEGF), leading to vascular leakage and hemorrhage. Angiopoietin 2 can act as an antagonist to Tie-2,
destabilizing the endothelial cells, which results in less endothelial
cell and pericyte interaction. This occasionally leads to the formation
of tumors. Similar to the inhibition of the PDGF pathway, angiopoietin 2 reduces levels of pericytes, leading to diabetic retinopathy.
Scarring
Usually, astrocytes are associated with the scarring process in the central nervous system, forming glial scars.
It has been proposed that a subtype of pericytes participates in this
scarring in a glial-independent manner. Through lineage tracking
studies, these subtype of pericytes were followed after stroke,
revealing that they contribute to the glial scar by differentiating into
myofibroblasts and depositing extracellular matrix.
However, this remains controversial, as more recent studies suggest
that the cell type followed in these scar studies is likely to be not
pericytes, but fibroblasts.
Contribution to adult neurogenesis
The
emerging evidence (as of 2019) suggests that neural microvascular
pericytes, under instruction from resident glial cells, are reprogrammed
into interneurons and enrich local neuronal microcircuits.[52] This response is amplified by concomitant angiogenesis.
The blood–brain barrier (BBB) is a highly selective semipermeable border of endothelial cells that regulates the transfer of solutes and chemicals between the circulatory system and the central nervous system, thus protecting the brain from harmful or unwanted substances in the blood. The blood–brain barrier is formed by endothelial cells of the capillary wall, astrocyte end-feet ensheathing the capillary, and pericytes embedded in the capillary basement membrane. This system allows the passage of some small molecules by passive diffusion,
as well as the selective and active transport of various nutrients,
ions, organic anions, and macromolecules such as glucose and amino acids that are crucial to neural function.
The blood–brain barrier restricts the passage of pathogens, the diffusion of solutes in the blood, and large or hydrophilic molecules into the cerebrospinal fluid, while allowing the diffusion of hydrophobic molecules (O2, CO2, hormones) and small non-polar molecules. Cells of the barrier actively transport metabolic products such as glucose across the barrier using specific transport proteins.
The barrier also restricts the passage of peripheral immune factors,
like signaling molecules, antibodies, and immune cells, into the CNS,
thus insulating the brain from damage due to peripheral immune events.
Part of a network of capillaries supplying brain cellsThe astrocytes type 1 surrounding capillaries in the brainSketch showing constitution of blood vessels inside the brain
The BBB results from the selectivity of the tight junctions between the endothelial cells of brain capillaries, restricting the passage of solutes.
At the interface between blood and the brain, endothelial cells are
adjoined continuously by these tight junctions, which are composed of
smaller subunits of transmembrane proteins, such as occludin, claudins (such as Claudin-5), junctional adhesion molecule (such as JAM-A).
Each of these tight junction proteins is stabilized to the endothelial
cell membrane by another protein complex that includes scaffolding
proteins such as tight junction protein 1 (ZO1) and associated proteins.
The BBB is composed of endothelial cells restricting passage of
substances from the blood more selectively than endothelial cells of
capillaries elsewhere in the body. Astrocyte cell projections called astrocytic feet (also known as "glia limitans") surround the endothelial cells of the BBB, providing biochemical support to those cells. The BBB is distinct from the quite similar blood-cerebrospinal fluid barrier, which is a function of the choroidal cells of the choroid plexus, and from the blood-retinal barrier, which can be considered a part of the whole realm of such barriers.
Not all vessels in the human brain exhibit BBB properties. Some examples of this include the circumventricular organs, the roof of the third and fourth ventricles, capillaries in the pineal gland on the roof of the diencephalon and the pineal gland. The pineal gland secretes the hormone melatonin "directly into the systemic circulation", thus melatonin is not affected by the blood–brain barrier.
Development
The BBB appears to be functional by the time of birth. P-glycoprotein, a transporter, exists already in the embryonal endothelium.
Measurement of brain uptake of various blood-borne solutes showed
that newborn endothelial cells were functionally similar to those in
adults, indicating that a selective BBB is operative at birth.
In mice, Claudin-5 loss during development is lethal and results in size-selective loosening of the BBB.
The blood–brain barrier acts effectively to protect brain tissue from circulating pathogens and other potentially toxic substances. Accordingly, blood-borne infections of the brain are rare. Infections of the brain that do occur are often difficult to treat. Antibodies are too large to cross the blood–brain barrier, and only certain antibiotics are able to pass.
In some cases, a drug has to be administered directly into the
cerebrospinal fluid where it can enter the brain by crossing the blood-cerebrospinal fluid barrier.
Permeable capillaries of the sensory CVOs (area postrema,
subfornical organ, vascular organ of the lamina terminalis) enable rapid
detection of circulating signals in systemic blood, while those of the
secretory CVOs (median eminence, pineal gland, pituitary lobes)
facilitate transport of brain-derived signals into the circulating
blood. Consequently, the CVO permeable capillaries are the point of bidirectional blood–brain communication for neuroendocrine function.
Specialized permeable zones
The
border zones between brain tissue "behind" the blood–brain barrier and
zones "open" to blood signals in certain CVOs contain specialized hybrid
capillaries that are leakier than typical brain capillaries, but not as
permeable as CVO capillaries. Such zones exist at the border of the
area postrema—nucleus tractus solitarii (NTS), and median eminence—hypothalamicarcuate nucleus.
These zones appear to function as rapid transit regions for brain
structures involved in diverse neural circuits—like the NTS and arcuate
nucleus—to receive blood signals which are then transmitted into neural
output.The permeable capillary zone shared between the median eminence and
hypothalamic arcuate nucleus is augmented by wide pericapillary spaces,
facilitating bidirectional flow of solutes between the two structures,
and indicating that the median eminence is not only a secretory organ,
but may also be a sensory organ.
Therapeutic research
As a drug target
The
blood–brain barrier is formed by the brain capillary endothelium and
excludes from the brain 100% of large-molecule neurotherapeutics and
more than 98% of all small-molecule drugs.
Overcoming the difficulty of delivering therapeutic agents to specific
regions of the brain presents a major challenge to treatment of most
brain disorders. In its neuroprotective role, the blood–brain barrier functions to
hinder the delivery of many potentially important diagnostic and
therapeutic agents to the brain. Therapeutic molecules and antibodies
that might otherwise be effective in diagnosis and therapy do not cross
the BBB in adequate amounts to be clinically effective.
The BBB represents an obstacle to some drugs reaching the brain, thus
to overcome this barrier some peptides able to naturally cross the BBB
have been widely investigated as a drug delivery system.
Other methods used to get through the BBB may entail the use of
endogenous transport systems, including carrier-mediated transporters,
such as glucose and amino acid carriers, receptor-mediated transcytosis for insulin or transferrin, and the blocking of active efflux transporters such as p-glycoprotein. Some studies have shown that vectors targeting BBB transporters, such as the transferrin receptor,
have been found to remain entrapped in brain endothelial cells of
capillaries, instead of being ferried across the BBB into the targeted
area.
Nanotechnology is under preliminary research for its potential to facilitate the transfer of drugs across the BBB.Capillary endothelial cells and associated pericytes may be abnormal in tumors and the blood–brain barrier may not always be intact in brain tumors. Other factors, such as astrocytes, may contribute to the resistance of brain tumors to therapy using nanoparticles. Fat soluble molecules less than 400 daltons in mass can freely diffuse past the BBB through lipid mediated passive diffusion.
There have been many attempts to correlate the experimental blood–brain barrier permeability with physicochemical properties. The first QSAR
study of brain–blood distribution was conducted in 1988, this study
reported the in vivo values in rats for a large number of H2 receptor
histamine agonists.
The first papers trying to model blood brain barrier permeability,
identified three properties, i.e., molecular volume, lipophilicity, and
hydrogen bonding potential, as contributing significantly to the
transport through the blood brain barrier.
Two datasets, one with numerical logBB values (1058 molecules) and the
one with categorical labels (7807 molecules with 4956 BBB+ and 2851
BBB−) have been published in 2021. The categorical dataset has been used in 2022 to select four different classification models based on molecular fingerprints, MACCS166 keys and molecular descriptors.
History
In 1898, Arthur Biedl and R. Kraus observed that low-concentration "bile salts" failed to affect behavior when injected into the bloodstream of animals. Thus, in theory, they had failed to enter the brain.
Two years later, Max Lewandowsky may have been the first to coin the term "blood–brain barrier" in 1900, referring to the hypothesized semipermeable membrane. There is some debate over the creation of the term blood–brain barrier as it is often attributed to Lewandowsky, but it does not appear in his papers. The creator of the term may have been Lina Stern.
Stern was a Russian scientist who published her work in Russian and
French. Due to the language barrier between her publications and
English-speaking scientists, this could have made her work a
lesser-known origin of the term.
All the while, bacteriologistPaul Ehrlich was studying staining, a procedure that is used in many microscopy studies to make fine biological structures visible using chemical dyes. As Ehrlich injected some of these dyes (notably the aniline dyes that were then widely used), the dye stained all of the organs of some kinds of animals except for their brains. At that time, Ehrlich attributed this lack of staining to the brain simply not picking up as much of the dye.
However, in a later experiment in 1913, Edwin Goldmann (one of Ehrlich's students) injected the dye directly into the cerebrospinal fluid
of animal brains. He found then the brains did become dyed, but the
rest of the body did not, demonstrating the existence of a
compartmentalization between the two. At that time, it was thought that
the blood vessels themselves were responsible for the barrier, since no obvious membrane could be found.
Drug delivery to the brain is the process of passing therapeutically active molecules across the blood–brain barrier into the brain.
This is a complex process that must take into account the complex
anatomy of the brain as well as the restrictions imposed by the special
junctions of the blood–brain barrier.
Anatomy
The blood–brain barrier is formed by special tight junctions between endothelial cells lining brain blood vessels. Blood vessels of all tissues contain this monolayer of endothelial cells, however only brain endothelial cells have tight junctions preventing passive diffusion of most substances into the brain tissue.
The structure of these tight junctions was first determined in the
1960s by Tom Reese, Morris Kranovsky, and Milton Brightman. Furthermore,
astrocytic "end feet", the terminal regions of the astrocytic processes, surround the outside of brain capillaryendothelial cells". The astrocytes are glial cells restricted to the brain and spinal cord and help maintain blood-brain barrier properties in brain endothelial cells.
Physiology
The
main function of the blood–brain barrier is to protect the brain and
keep it isolated from harmful toxins that are potentially in the blood stream. It accomplishes this because of its structure, as is usual in the body that structure defines its function. The tight junctions between the endothelial cells prevent large molecules as well as many ions from passing between the junction spaces. This forces molecules to go through the endothelial cells in order to enter the brain tissue, meaning that they must pass through the cell membranes of the endothelial cells.
Because of this, the only molecules that are easily able to transverse the blood–brain barrier are ones that are very lipid-soluble. These are not the only molecules that can transverse the blood–brain barrier; glucose, oxygen and carbon dioxide are not lipid-soluble but are actively transported across the barrier, to support normal cellular function of the brain. The fact that molecules have to fully transverse the endothelial cells
makes them a perfect barricade to unspecified particles from entering
the brain, working to protect the brain at all costs. Also, because most
molecules are transported across the barrier, it does a very effective
job of maintaining homeostasis for the most vital organ of the human body.
Drug delivery to the blood–brain barrier
Because of the difficulty for drugs
to pass through the blood–brain barrier, a study was conducted to
determine the factors that influence a compound’s ability to transverse
the blood–brain barrier. In this study, they examined several different
factors to investigate diffusion across the blood–brain barrier. They used lipophilicity, Gibbs Adsorption Isotherm, a Co CMC Plot, and the surface area of the drug to water and air. They began by looking at compounds whose blood–brain permeability was known and labeled them either CNS+ or CNS- for compounds that easily transverse the barrier and those that did not.
They then set out to analyze the above factors to determine what is
necessary to transverse the blood–brain barrier. What they found was a
little surprising; lipophilicity
is not the leading characteristic for a drug to pass through the
barrier. This is surprising because one would think that the most
effective way to make a drug move through a lipophilic barrier is to increase its lipophilicity,
it turns out that it is a complex function of all of these
characteristics that makes a drug able to pass through the blood–brain
barrier. The study found that barrier permittivity is "based on the measurement of the surface activity and as such takes into account the molecular properties of both hydrophobic and charged residues of the molecule of interest."
They found that there is not a simple answer to what compounds
transverse the blood–brain barrier and what does not. Rather, it is
based on the complex analysis of the surface activity of the molecule as
well as relative size.
Problems faced in drug delivery
Other
problems persist besides just simply getting through the blood–brain
barrier. The first of these is that a lot of times, even if a compound
transverses the barrier, it does not do it in a way that the drug is in a therapeutically relevant concentration.
This can have many causes, the most simple being that the way the drug
was produced only allows a small amount to pass through the barrier.
Another cause of this would be the binding to other proteins
in the body rendering the drug ineffective to either be therapeutically
active or able to pass through the barrier with the adhered protein. Another problem that must be accounted for is the presence of enzymes in the brain tissue
that could render the drug inactive. The drug may be able to pass
through the membrane fine, but will be deconstructed once it is inside
the brain tissue rendering it useless. All of these are problems that
must be addressed and accounted for in trying to deliver effective drug
solutions to the brain tissue.
Possible solutions
Exosomes to deliver treatments across the blood–brain barrier
A group from the University of Oxford led by Prof. Matthew Wood claims that exosomes can cross the blood–brain barrier and deliver siRNAs,
antisense oligonucleotides, chemotherapeutic agents and proteins
specifically to neurons after inject them systemically (in blood).
Because these exosomes are able to cross the blood–brain barrier, this
protocol could solve the issue of poor delivery of medications to the
central nervous system and cure Alzheimer's, Parkinson's Disease and
brain cancer, among other diseases. The laboratory has been recently
awarded a major new €30 million project leading experts from 14 academic
institutions, two biotechnology companies and seven pharmaceutical
companies to translate the concept to the clinic.
Pro-drugs
This is the process of disguising medically active molecules with lipophilic molecules that allow it to better sneak through the blood–brain barrier. Drugs can be disguised using more lipophilic elements or structures. This form of the drug will be inactive because of the lipophilic molecules but then would be activated, by either enzyme degradation or some other mechanism for removal of the lipophilic
disguise to release the drug into its active form. There are still some
major drawbacks to these pro-drugs. The first of which is that the
pro-drug may be able to pass through the barrier and then also re-pass
through the barrier without ever releasing the drug in its active form.
The second is the sheer size of these types of molecules makes it still
difficult to pass through the blood–brain barrier.
Peptide masking
Similar to the idea of pro-drugs, another way of masking the drugs chemical composition is by masking a peptide’s
characteristics by combining with other molecular groups that are more
likely to pass through the blood–brain barrier. An example of this is
using a cholesteryl molecule instead of cholesterol that serves to conceal the water soluble
characteristics of the drug. This type of masking as well as aiding in
traversing the blood–brain barrier. It also can work to mask the drug
peptide from peptide-degrading enzymes in the brain
Also a "targetor" molecule could be attached to the drug that helps it
pass through the barrier and then once inside the brain, is degraded in
such a way that the drug cannot pass back through the brain. Once the
drug cannot pass back through the barrier the drug can be concentrated
and made effective for therapeutic use.
However drawbacks to this exist as well. Once the drug is in the brain
there is a point where it needs to be degraded to prevent overdose to the brain tissue.
Also if the drug cannot pass back through the blood–brain barrier, it
compounds the issues of dosage and intense monitoring would be required.
For this to be effective there must be a mechanism for the removal of
the active form of the drug from the brain tissue.
Receptor-mediated permabilitizers
These are drug compounds that increase the permeability of the blood–brain barrier.
By decreasing the restrictiveness of the barrier, it is much easier to
get a molecule to pass through it. These drugs increase the permeability of the blood–brain barrier temporarily by increasing the osmotic pressure in the blood which loosens the tight junctions between the endothelial cells. By loosening the tight junctions normal injection of drugs through an [IV] can take place and be effective to enter the brain.
This must be done in a very controlled environment because of the risk
associated with these drugs. Firstly, the brain can be flooded with
molecules that are floating through the blood stream that are usually blocked by the barrier. Secondly, when the tight junctions loosen, the homeostasis of the brain can also be thrown off which can result in seizures and the compromised function of the brain.
Nanoparticles
The most promising drug delivery system is using nanoparticle
delivery systems, these are systems where the drug is bound to a
nanoparticle capable of traversing the blood–brain barrier. The most
promising compound for the nanoparticles is Human Serum Albumin (HSA). The main benefits of this is that particles made of HSA are well tolerated without serious side effects as well as the albumin functional groups can be utilized for surface modification that allows for specific cell uptake. These nanoparticles
have been shown to transverse the blood–brain barrier carrying host
drugs. To enhance the effectiveness of nanoparticles, scientists are
attempting to coat the nanoparticles
to make them more effective to cross the blood–brain barrier. Studies
have shown that "the overcoating of the [nanoparticles] with polysorbate
80 yielded doxorubicin concentrations in the brain of up to 6 μg/g
after i.v. injection of 5 mg/kg" as compared to no detectable increase
in an injection of the drug alone or the uncoated nanoparticle.
This is very new science and technology so the real effectiveness of
this process has not been fully understood. However young the research
is, the results are promising pointing to nanotechnology as the way forward in treating a variety of brain diseases.
Loaded microbubble-enhanced focused ultrasound
Microbubbles are small "bubbles" of mono-lipids that are able to pass through the blood–brain barrier. They form a lipophilic bubble that can easily move through the barrier. One barrier to this however is that these microbubbles are rather large, which prevents their diffusion into the brain. This is counteracted by a focused ultrasound. The ultrasound increases the permeability of the blood–brain barrier by causing interference in the tight junctions in localized areas. This combined with the microbubbles allows for a very specific area of diffusion for the microbubbles, because they can only diffuse where the ultrasound is disrupting the barrier. The hypothesis and usefulness of these is the possibility of loading a microbubble with an active drug to diffuse through the barrier and target a specific area. There are several important factors in making this a viable solution for drug delivery. The first is that the loaded microbubble must not be substantially greater than the unloaded bubble. This ensures that the diffusion will be similar and the ultrasound disruption will be enough to induce diffusion.
A second factor that must be determined is the stability of the loaded
micro-bubble. This means is the drug fully retained in the bubble or is
there leakage. Lastly, it must be determined how the drug is to be
released from the microbubble
once it passes through the blood–brain barrier. Studies have shown the
effectiveness of this method for getting drugs to specific sites in the
brain in animal models.
An artificial cell, synthetic cell or minimal cell is an engineered particle that mimics one or many functions of a biological cell. Often, artificial cells are biological or polymeric membranes which enclose biologically active materials. As such, liposomes, polymersomes, nanoparticles, microcapsules and a number of other particles can qualify as artificial cells.
The terms "artificial cell" and "synthetic cell" are used in a
variety of different fields and can have different meanings, as it is
also reflected in the different sections of this article. Some stricter
definitions are based on the assumption that the term "cell" directly
relates to biological cells
and that these structures therefore have to be alive (or part of a
living organism) and, further, that the term "artificial" implies that
these structures are artificially built from the bottom-up, i.e. from
basic components. As such, in the area of synthetic biology, an artificial cell can be understood as a completely synthetically made cell that can capture energy, maintain ion gradients, contain macromolecules as well as store information and have the ability to replicate. This kind of artificial cell has not yet been made.
However, in other cases, the term "artificial" does not imply
that the entire structure is man-made, but instead, it can refer to the
idea that certain functions or structures of biological cells can be
modified, simplified, replaced or supplemented with a synthetic entity.
In other fields, the term "artificial cell" can refer to any
compartment that somewhat resembles a biological cell in size or
structure, but is synthetically made, or even fully made from
non-biological components. The term "artificial cell" is also used for
structures with direct applications such as compartments for drug
delivery. Micro-encapsulation allows for metabolism within the membrane, exchange of small molecules and prevention of passage of large substances across it. The main advantages of encapsulation include improved mimicry in the body, increased solubility of the cargo and decreased immune responses. Notably, artificial cells have been clinically successful in hemoperfusion.
Bottom-up engineering of living artificial cells
Diagram of lipid vesicles showing a solution of biomolecules (green dots) trapped in the vesicle interior.
The German pathologist Rudolf Virchow brought forward the idea that not only does life arise from cells, but every cell comes from another cell; "Omnis cellula e cellula".
Until now, most attempts to create an artificial cell have only created
a package that can mimic certain tasks of the cell. Advances in
cell-free transcription and translation reactions allow the expression of many genes, but these efforts are far from producing a fully operational cell.
A bottom-up approach to build an artificial cell would involve creating a protocellde novo,
entirely from non-living materials. As the term "cell" implies, one
prerequisite is the generation of some sort of compartment that defines
an individual, cellular unit. Phospholipid membranes are an obvious choice as compartmentalizing boundaries,
as they act as selective barriers in all living biological cells.
Scientists can encapsulate biomolecules in cell-sized phospholipid vesicles and by doing so, observe these molecules to act similarly as in biological cells and thereby recreate certain cell functions.
In a similar way, functional biological building blocks can be
encapsulated in these lipid compartments to achieve the synthesis of
(however rudimentary) artificial cells.
It is proposed to create a phospholipid bilayer vesicle with DNA
capable of self-reproducing using synthetic genetic information. The
three primary elements of such artificial cells are the formation of a lipid membrane, DNA and RNA replication through a template process and the harvesting of chemical energy for active transport across the membrane.
The main hurdles foreseen and encountered with this proposed protocell
are the creation of a minimal synthetic DNA that holds all sufficient
information for life, and the reproduction of non-genetic components
that are integral in cell development such as molecular
self-organization.
However, it is hoped that this kind of bottom-up approach would provide
insight into the fundamental questions of organizations at the cellular
level and the origins of biological life. So far, no completely
artificial cell capable of self-reproduction has been synthesized using
the molecules of life, and this objective is still in a distant future
although various groups are currently working towards this goal.
Another method proposed to create a protocell more closely resembles the conditions
believed to have been present during evolution known as the primordial
soup. Various RNA polymers could be encapsulated in vesicles and in such
small boundary conditions, chemical reactions would be tested for.
Ethics and controversy
Protocell
research has created controversy and opposing opinions, including
critics of the vague definition of "artificial life". The creation of a basic unit of life is the most pressing ethical concern.
The most widespread worry about protocells is their potential threat to
human health and the environment through uncontrolled replication.
However, artificial cells made through a top-down approach, or any other
manipulated forms of existing living cells, are much more likely to be
able to exist and reproduce outside of a laboratory and therefore to
pose such a threat.
International Research Community
In
the mid-2010s the research community started recognising the need to
unify the field of synthetic cell research, acknowledging that the task
of constructing an entire living organism from non-living components was
beyond the resources of a single country.
In 2017 the NSF-funded international Build-a-Cell large-scale research collaboration for the construction of synthetic living cell was started,.
Build-a-Cell has conducted nine interdisciplinary workshopping events,
open to all interested, to discuss and guide the future of the synthetic
cell community. Build-a-Cell was followed by national synthetic cell
organizations in several other countries. Those national organizations
include FabriCell, MaxSynBio and BaSyC. The European synthetic cell efforts were unified in 2019 as SynCellEU initiative.
Top-down approach to create a minimal living cell
Members from the J. Craig Venter Institute have used a top-down computational approach to knock out genes in a living organism to a minimum set of genes. In 2010, the team succeeded in creating a replicating strain (named Mycoplasma laboratorium) of Mycoplasma mycoides
using synthetically created DNA deemed to be the minimum requirement
for life which was inserted into a genomically empty bacterium.
It is hoped that the process of top-down biosynthesis will enable the
insertion of new genes that would perform profitable functions such as
generation of hydrogen for fuel or capturing excess carbon dioxide in
the atmosphere. The myriad regulatory, metabolic, and signaling networks are not completely characterized. These top-down
approaches have limitations for the understanding of fundamental
molecular regulation, since the host organisms have a complex and
incompletely defined molecular composition.
In 2019 a complete computational model of all pathways in Mycoplasma
Syn3.0 cell was published, representing the first complete in silico model for a living minimal organism.
Heavy investing in biology has been done by large companies such as ExxonMobil, who has partnered with Synthetic Genomics Inc; Craig Venter's own biosynthetics company in the development of fuel from algae.
As of 2016, Mycoplasma genitalium
is the only organism used as a starting point for engineering a minimal
cell, since it has the smallest known genome that can be cultivated
under laboratory conditions; the wild-type variety has 482, and removing
exactly 100 genes deemed non-essential resulted in a viable strain with
improved growth rates. Reduced-genome Escherichia coli is considered more useful, and viable strains have been developed with 15% of the genome removed.
A variation of an artificial cell has been created in which a completely synthetic genome was introduced to genomically emptied host cells. Although not completely artificial because the cytoplasmic components as well as the membrane from the host cell are kept, the engineered cell is under control of a synthetic genome and is able to replicate.
Artificial cells for medical applications
Standard artificial cell (top) and drug delivery artificial cell (bottom).
History
In the 1960s Thomas Chang
developed microcapsules which he would later call "artificial cells",
as they were cell-sized compartments made from artificial materials. These cells consisted of ultrathin membranes of nylon, collodion or crosslinked protein whose semipermeable properties allowed diffusion of small molecules in and out of the cell. These cells were micron-sized and contained cells, enzymes, hemoglobin, magnetic materials, adsorbents and proteins.
Later artificial cells have ranged from hundred-micrometer to nanometer dimensions and can carry microorganisms, vaccines, genes, drugs, hormones and peptides. The first clinical use of artificial cells was in hemoperfusion by the encapsulation of activated charcoal.
In the 1970s, researchers were able to introduce enzymes,
proteins and hormones to biodegradable microcapsules, later leading to
clinical use in diseases such as Lesch–Nyhan syndrome. Although Chang's initial research focused on artificial red blood cells, only in the mid-1990s were biodegradable artificial red blood cells developed. Artificial cells in biological cell encapsulation were first used in the clinic in 1994 for treatment in a diabetic patient and since then other types of cells such as hepatocytes, adult stem cells and genetically engineered cells have been encapsulated and are under study for use in tissue regeneration.
Materials
Representative types of artificial cell membranes.
Membranes for artificial cells can be made of simple polymers, crosslinked proteins, lipid membranes or polymer-lipid complexes. Further, membranes can be engineered to present surface proteins such as albumin, antigens, Na/K-ATPase carriers, or pores such as ion channels. Commonly used materials for the production of membranes include hydrogel polymers such as alginate, cellulose and thermoplastic
polymers such as hydroxyethyl methacrylate-methyl methacrylate (HEMA-
MMA), polyacrylonitrile-polyvinyl chloride (PAN-PVC), as well as
variations of the above-mentioned.
The material used determines the permeability of the cell membrane,
which for polymer depends on the is important in determining adequate diffusion of nutrients, waste and other critical molecules. Hydrophilic polymers have the potential to be biocompatible and can be fabricated into a variety of forms which include polymer micelles, sol-gel mixtures, physical blends and crosslinked particles and nanoparticles. Of special interest are stimuli-responsive polymers that respond to pH
or temperature changes for the use in targeted delivery. These polymers
may be administered in the liquid form through a macroscopic injection
and solidify or gel in situ because of the difference in pH or temperature. Nanoparticle and liposome
preparations are also routinely used for material encapsulation and
delivery. A major advantage of liposomes is their ability to fuse to cell and organelle membranes.
Preparation
Many variations for artificial cell preparation and encapsulation have been developed. Typically, vesicles such as a nanoparticle, polymersome or liposome are synthesized. An emulsion is typically made through the use of high pressure equipment such as a high pressure homogenizer or a Microfluidizer. Two micro-encapsulation methods for nitrocellulose are also described below.
High-pressure homogenization
In
a high-pressure homogenizer, two liquids in oil/liquid suspension are
forced through a small orifice under very high pressure. This process
divides the products and allows the creation of extremely fine
particles, as small as 1 nm.
Microfluidization
This
technique uses a patented Microfluidizer to obtain a greater amount of
homogenous suspensions that can create smaller particles than
homogenizers. A homogenizer is first used to create a coarse suspension
which is then pumped into the microfluidizer under high pressure. The
flow is then split into two streams which will react at very high
velocities in an interaction chamber until desired particle size is
obtained. This technique allows for large scale production of phospholipid liposomes and subsequent material nanoencapsulations.
Drop method
In this method, a cell solution is incorporated dropwise into a collodion
solution of cellulose nitrate. As the drop travels through the
collodion, it is coated with a membrane thanks to the interfacial
polymerization properties of the collodion. The cell later settles into
paraffin, where the membrane sets, which is then suspended using a
saline solution. The drop method is used for the creation of large
artificial cells which encapsulate biological cells, stem cells and
genetically engineered stem cells.
Emulsion method
The emulsion
method differs in that the material to be encapsulated is usually
smaller and is placed in the bottom of a reaction chamber where the
collodion is added on top and centrifuged, or otherwise disturbed in
order to create an emulsion. The encapsulated material is then dispersed
and suspended in saline solution.
Clinical relevance
Drug release and delivery
Artificial cells used for drug delivery
differ from other artificial cells since their contents are intended to
diffuse out of the membrane, or be engulfed and digested by a host
target cell. Often used are submicron, lipid membrane artificial cells
that may be referred to as nanocapsules, nanoparticles, polymersomes, or
other variations of the term.
Enzyme therapy
Enzyme therapy is being actively studied for genetic metabolic diseases
where an enzyme is over-expressed, under-expressed, defective, or not
at all there. In the case of under-expression or expression of a
defective enzyme,
an active form of the enzyme is introduced in the body to compensate
for the deficit. On the other hand, an enzymatic over-expression may be
counteracted by introduction of a competing non-functional enzyme; that
is, an enzyme which metabolizes
the substrate into non-active products. When placed within an
artificial cell, enzymes can carry out their function for a much longer
period compared to free enzymes and can be further optimized by polymer conjugation.
The first enzyme studied under artificial cell encapsulation was asparaginase for the treatment of lymphosarcoma in mice. This treatment delayed the onset and growth of the tumor. These initial findings led to further research in the use of artificial cells for enzyme delivery in tyrosine dependent melanomas. These tumors have a higher dependency on tyrosine
than normal cells for growth, and research has shown that lowering
systemic levels of tyrosine in mice can inhibit growth of melanomas. The use of artificial cells in the delivery of tyrosinase;
and enzyme that digests tyrosine, allows for better enzyme stability
and is shown effective in the removal of tyrosine without the severe
side-effects associated with tyrosine depravation in the diet.
Artificial cell enzyme therapy is also of interest for the activation of prodrugs such as ifosfamide in certain cancers. Artificial cells encapsulating the cytochrome p450
enzyme which converts this prodrug into the active drug can be tailored
to accumulate in the pancreatic carcinoma or implanting the artificial
cells close to the tumor site. Here, the local concentration of the
activated ifosfamide will be much higher than in the rest of the body
thus preventing systemic toxicity. The treatment was successful in animals[42] and showed a doubling in median survivals amongst patients with advanced-stage pancreatic cancer in phase I/II clinical trials, and a tripling in one-year survival rate.
Gene therapy
In treatment of genetic diseases, gene therapy aims to insert, alter or remove genes within an afflicted individual's cells. The technology relies heavily on viral vectors which raises concerns about insertional mutagenesis and systemic immune response that have led to human deaths and development of leukemia
in clinical trials. Circumventing the need for vectors by using naked
or plasmid DNA as its own delivery system also encounters problems such
as low transduction efficiency and poor tissue targeting when given systemically.
Artificial cells have been proposed as a non-viral vector by
which genetically modified non-autologous cells are encapsulated and
implanted to deliver recombinant proteins in vivo. This type of immuno-isolation has been proven efficient in mice through delivery of artificial cells containing mouse growth hormone which rescued a growth-retardation in mutant mice. A few strategies have advanced to human clinical trials for the treatment of pancreatic cancer, lateral sclerosis and pain control.
Hemoperfusion
The first clinical use of artificial cells was in hemoperfusion by the encapsulation of activated charcoal.
Activated charcoal has the capability of adsorbing many large molecules
and has for a long time been known for its ability to remove toxic
substances from the blood in accidental poisoning or overdose. However, perfusion through direct charcoal administration is toxic as it leads to embolisms and damage of blood cells followed by removal by platelets. Artificial cells allow toxins to diffuse into the cell while keeping the dangerous cargo within their ultrathin membrane.
Artificial cell hemoperfusion has been proposed as a less costly and more efficient detoxifying option than hemodialysis,
in which blood filtering takes place only through size separation by a
physical membrane. In hemoperfusion, thousands of adsorbent artificial
cells are retained inside a small container through the use of two
screens on either end through which patient blood perfuses. As the blood circulates, toxins
or drugs diffuse into the cells and are retained by the absorbing
material. The membranes of artificial cells are much thinner those used
in dialysis and their small size means that they have a high membrane surface area.
This means that a portion of cell can have a theoretical mass transfer
that is a hundredfold higher than that of a whole artificial kidney
machine.
The device has been established as a routine clinical method for
patients treated for accidental or suicidal poisoning but has also been
introduced as therapy in liver failure and kidney failure by carrying out part of the function of these organs.
Artificial cell hemoperfusion has also been proposed for use in
immunoadsorption through which antibodies can be removed from the body
by attaching an immunoadsorbing material such as albumin on the surface of the artificial cells. This principle has been used to remove blood group antibodies from plasma for bone marrow transplantation and for the treatment of hypercholesterolemia through monoclonal antibodies to remove low-density lipoproteins.
Hemoperfusion is especially useful in countries with a weak
hemodialysis manufacturing industry as the devices tend to be cheaper
there and used in kidney failure patients.
Encapsulated cells
Schematic representation of encapsulated cells within artificial membrane.
The most common method of preparation of artificial cells is through cell encapsulation. Encapsulated cells are typically achieved through the generation of controlled-size droplets from a liquid cell suspension
which are then rapidly solidified or gelated to provide added
stability. The stabilization may be achieved through a change in
temperature or via material crosslinking. The microenvironment that a cell sees changes upon encapsulation. It typically goes from being on a monolayer
to a suspension in a polymer scaffold within a polymeric membrane. A
drawback of the technique is that encapsulating a cell decreases its
viability and ability to proliferate and differentiate. Further, after some time within the microcapsule, cells form clusters that inhibit the exchange of oxygen and metabolic waste, leading to apoptosis and necrosis thus limiting the efficacy of the cells and activating the host's immune system.
Artificial cells have been successful for transplanting a number of cells including islets of Langerhans for diabetes treatment, parathyroid cells and adrenal cortex cells.
Encapsulated hepatocytes
Shortage of organ donors make artificial cells key players in alternative therapies for liver failure. The use of artificial cells for hepatocyte transplantation has demonstrated feasibility and efficacy in providing liver function in models of animal liver disease and bioartificial liver devices. Research stemmed off experiments in which the hepatocytes were attached to the surface of a micro-carriers and has evolved into hepatocytes which are encapsulated in a three-dimensional matrix in alginate microdroplets covered by an outer skin of polylysine. A key advantage to this delivery method is the circumvention of immunosuppression therapy for the duration of the treatment. Hepatocyte encapsulations have been proposed for use in a bioartificial liver.
The device consists of a cylindrical chamber imbedded with isolated
hepatocytes through which patient plasma is circulated extra-corporeally
in a type of hemoperfusion. Because microcapsules have a high surface area to volume
ratio, they provide large surface for substrate diffusion and can
accommodate a large number of hepatocytes. Treatment to induced liver
failure mice showed a significant increase in the rate of survival. Artificial liver systems are still in early development but show potential for patients waiting for organ transplant
or while a patient's own liver regenerates sufficiently to resume
normal function. So far, clinical trials using artificial liver systems
and hepatocyte transplantation in end-stage liver diseases have shown
improvement of health markers but have not yet improved survival. The short longevity and aggregation of artificial hepatocytes after transplantation are the main obstacles encountered.
Hepatocytes co-encapsulated with stem cells show greater viability in culture and after implantation and implantation of artificial stem cells alone have also shown liver regeneration. As such interest has arisen in the use of stem cells for encapsulation in regenerative medicine.
Encapsulated bacterial cells
The oral ingestion of live bacterial cell colonies has been proposed and is currently in therapy for the modulation of intestinal microflora, prevention of diarrheal diseases, treatment of H. Pylori infections, atopic inflammations, lactose intolerance and immune modulation,
amongst others. The proposed mechanism of action is not fully
understood but is believed to have two main effects. The first is the
nutritional effect, in which the bacteria compete with toxin producing
bacteria. The second is the sanitary effect, which stimulates resistance
to colonization and stimulates immune response.
The oral delivery of bacterial cultures is often a problem because
they are targeted by the immune system and often destroyed when taken
orally. Artificial cells help address these issues by providing mimicry
into the body and selective or long term release thus increasing the
viability of bacteria reaching the gastrointestinal system.
In addition, live bacterial cell encapsulation can be engineered to
allow diffusion of small molecules including peptides into the body for
therapeutic purposes. Membranes that have proven successful for bacterial delivery include cellulose acetate and variants of alginate. Additional uses that have arosen from encapsulation of bacterial cells include protection against challenge from M. Tuberculosis and upregulation of Ig secreting cells from the immune system. The technology is limited by the risk of systemic infections, adverse metabolic activities and the risk of gene transfer. However, the greater challenge remains the delivery of sufficient viable bacteria to the site of interest.
Nano sized oxygen carriers are used as a type of red blood cell substitutes, although they lack other components of red blood cells. They are composed of a synthetic polymersome or an artificial membrane surrounding purified animal, human or recombinant hemoglobin.
Overall, hemoglobin delivery continues to be a challenge because it is
highly toxic when delivered without any modifications. In some clinical
trials, vasopressor effects have been observed.
Research interest in the use of artificial cells for blood arose after the AIDS
scare of the 1980s. Besides bypassing the potential for disease
transmission, artificial red blood cells are desired because they
eliminate drawbacks associated with allogenic blood transfusions such as
blood typing, immune reactions and its short storage life of 42 days. A
hemoglobin substitute may be stored at room temperature and not under refrigeration for more than a year.
Attempts have been made to develop a complete working red blood cell
which comprises carbonic not only an oxygen carrier but also the enzymes
associated with the cell. The first attempt was made in 1957 by
replacing the red blood cell membrane by an ultrathin polymeric membrane which was followed by encapsulation through a lipid membrane and more recently a biodegradable polymeric membrane.
A biological red blood cell membrane including lipids and associated proteins can also be used to encapsulate nanoparticles and increase residence time in vivo by bypassing macrophage uptake and systemic clearance.
Artificial leuko-polymersomes
A leuko-polymersome is a polymersome engineered to have the adhesive properties of a leukocyte. Polymersomes are vesicles composed of a bilayer sheet that can encapsulate many active molecules such as drugs or enzymes.
By adding the adhesive properties of a leukocyte to their membranes,
they can be made to slow down, or roll along epithelial walls within the
quickly flowing circulatory system.
Unconventional types of artificial cells
Electronic artificial cell
The
concept of an Electronic Artificial Cell has been expanded in a series
of 3 EU projects coordinated by John McCaskill from 2004 to 2015.
The European Commission sponsored the development of the Programmable Artificial Cell Evolution (PACE) program
from 2004 to 2008 whose goal was to lay the foundation for the creation
of "microscopic self-organizing, self-replicating, and evolvable
autonomous entities built from simple organic and inorganic substances
that can be genetically programmed to perform specific functions"
for the eventual integration into information systems. The PACE project
developed the first Omega Machine, a microfluidic life support system
for artificial cells that could complement chemically missing
functionalities (as originally proposed by Norman Packard, Steen
Rasmussen, Mark Beadau and John McCaskill). The ultimate aim was to
attain an evolvable hybrid cell in a complex microscale programmable
environment. The functions of the Omega Machine could then be removed
stepwise, posing a series of solvable evolution challenges to the
artificial cell chemistry. The project achieved chemical integration up
to the level of pairs of the three core functions of artificial cells (a
genetic subsystem, a containment system and a metabolic system), and
generated novel spatially resolved programmable microfluidic
environments for the integration of containment and genetic
amplification. The project led to the creation of the European center for living technology.
Following this research, in 2007, John McCaskill proposed to
concentrate on an electronically complemented artificial cell, called
the Electronic Chemical Cell. The key idea was to use a massively
parallel array of electrodes coupled to locally dedicated electronic
circuitry, in a two-dimensional thin film, to complement emerging
chemical cellular functionality. Local electronic information defining
the electrode switching and sensing circuits could serve as an
electronic genome, complementing the molecular sequential information in
the emerging protocols. A research proposal was successful with the European Commission
and an international team of scientists partially overlapping with the
PACE consortium commenced work 2008–2012 on the project Electronic
Chemical Cells. The project demonstrated among other things that
electronically controlled local transport of specific sequences could be
used as an artificial spatial control system for the genetic
proliferation of future artificial cells, and that core processes of
metabolism could be delivered by suitably coated electrode arrays.
The major limitation of this approach, apart from the initial
difficulties in mastering microscale electrochemistry and
electrokinetics, is that the electronic system is interconnected as a
rigid non-autonomous piece of macroscopic hardware. In 2011, McCaskill
proposed to invert the geometry of electronics and chemistry : instead
of placing chemicals in an active electronic medium, to place
microscopic autonomous electronics in a chemical medium. He organized a
project to tackle a third generation of Electronic Artificial Cells at
the 100 µm scale that could self-assemble from two half-cells "lablets"
to enclose an internal chemical space, and function with the aid of
active electronics powered by the medium they are immersed in. Such
cells can copy both their electronic and chemical contents and will be
capable of evolution within the constraints provided by their special
pre-synthesized microscopic building blocks. In September 2012 work
commenced on this project.
Artificial neurons
There is research and development into physical artificial neurons – organic and inorganic.
Organic neuromorphic circuits made out of polymers, coated with an ion-rich gel to enable a material to carry an electric charge like real neurons,
have been built into a robot, enabling it to learn sensorimotorically
within the real world, rather than via simulations or virtually.
Moreover, artificial spiking neurons made of soft matter (polymers) can
operate in biologically relevant environments and enable the synergetic
communication between the artificial and biological domains.
Jeewanu protocells are synthetic chemical particles that possess cell-like structure and seem to have some functional living properties. First synthesized in 1963 from simple minerals and basic organics while exposed to sunlight, it is still reported to have some metabolic capabilities, the presence of semipermeable membrane, amino acids, phospholipids, carbohydrates and RNA-like molecules. However, the nature and properties of the Jeewanu remains to be clarified.
Semi-artificial cyborg cells
A combination of synthetic biology, nanotechnology and materials science approaches have been used to create a few different iterations of bacterial cyborg cells. These different types of mechanically enhanced bacteria are created with so called bionic manufacturing
principles that combine natural cells with abiotic materials. In 2005,
researchers from the Department of Chemical Engineering at the University of Nebraska, Lincoln created a super sensitive humidity sensor by coating the bacteria Bacillus cereus
with gold nanoparticles, being the first to use a microorganism to make
an electronic device and presumably the first cyborg bacteria or
cellborg circuit. Researchers from the Department of Chemistry at the University of California, Berkeley
published a series of articles in 2016 describing the development of
cyborg bacteria capable to harvest sunlight more efficiently than
plants. In the first study, the researchers induced the self-photosensitization of a nonphotosynthetic bacterium, Moorella thermoacetica, with cadmium sulfide nanoparticles, enabling the photosynthesis of acetic acid from carbon dioxide.
A follow-up article described the elucidation of the mechanism of
semiconductor-to-bacterium electron transfer that allows the
transformation of carbon dioxide and sunlight into acetic acid. Scientists of the Department of Biomedical Engineering at the University of California, Davis and Academia Sinica in Taiwan, developed a different approach to create cyborg cells by assembling a synthetic hydrogel inside the bacterial cytoplasm of Escherichia. coli cells rendering them incapable of dividing and making them resistant to environmental factors, antibiotics and high oxidative stress. The intracellular infusion of synthetic hydrogel provides these cyborg cells with an artificial cytoskeleton and their acquired tolerance makes them well placed to become a new class of drug-delivery systems positioned between classical synthetic materials and cell-based systems.
