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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5373_Библиотеки_им_академика_М_И_Перельмана.pdf
14 Reversible PEGylation ofNanocarriers
407
14.3.2.6 Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA can be effectively used for the measurement of the concentration of the
PEGylated proteins in serum samples. This technique provides an idea about the
effect of PEGylation on the immunogenicity of the proteins. In this, a specic antigen is coated with microliter plate wells to capture PEGylated antibodies in the
sample, and then, the amount of antibody bound to the antigen is determined by
adding a detection antibody. A direct ELISA methodology using the antigen-coated
microliter plates can also be used for the pharmacokinetics determination of the
PEGylated nanocarriers. By using the competition assay methodology, direct
ELISA can be used to measure the concentration of PEGylated compounds. In this
case, the PEGylated nanocarrier is coated in the wells of a microtiter plate and the
serum concentration of the PEGylated nanocarrier can be estimated from the degree
of binding of the specic antibody to the plate coated with the compound (Filpula
and Zhao 2008).
14.3.2.7 Sandwich ELISA
As the simple ELISA methodology requires only one specic antibody for the
detection of PEGylated compound, it cannot usually differentiate between the
PEGylate and non-PEGylated proteins or nanocarriers. So, the sophisticated methodologies are currently used, for example, the indirect or Sandwich ELISA which
makes the use of two antibodies, one to capture the analyte on the solid surface and
the other to effectively determine the concentration of the detected analyte. In order
to get detected, the analyte must possess at least two antigen-binding sites, that can
be considered as the distinct binding epitopes present on the same analyte. For multimeric analytes, the same epitope can be used as the detection antibodies that bind
to the same molecule. Sandwich ELISA is used to measure the concentration of the
PEGylated nanocarriers in the complex biological samples. This is a more sensitive
technique than direct and competitive ELISA and often considered with due priority
when the analyte is present in low concentration (Liu etal. 2020).
14.3.2.8 Anti-PEG ELISA
ELISA can be made more accurate and sensitive when the antibodies that specically bind to PEG are used. For the generation of PEG-specic antibodies, a laboratory animal is infected with PEG linked proteins. The polyclonal antibodies thus
obtained can be effectively employed for the specic binding of PEGylated nanocarriers. As the PEG possess very low immunogenicity, the animal is exposed to the
PEG attached protein multiple times to attain the required extent of immunogenicity
(Ozer etal. 2022).
14.3.3 In Vivo Characterization ofReversible PEGylation
14.3.3.1 In Vivo Blood Circulation Half-Life
The pharmacokinetic and tissue distribution studies are the popular kinds of invivo
assessment for the PEGylated nanocarriers. If the PEGylation is achieved to the
408
M. Mishra et al.
sufcient extent, it should increase the circulation time of the formulation in blood.
It should also cause the reduction in the uptake by the liver as compared to the nonPEGylated particles (Howard etal. 2008). The main purpose of the PEGylation of
the nanocarriers is to increase their residence time inside the body. The circulation
half-life of the PEGylated nanoparticles can be measured using mice as an animal model.
In the estimation, it is evident that the nanoparticles having lower PEG surface
coverage will have lowest circulation time and the blood circulation time will then
increase as the concentration of PEG on surface is increased. The circulation halflife was determined in female BALB/c nude mice. Mice aged 6weeks are housed
into four groups in different cages, while maintaining the temperature and relative
humidity. The animals were then anesthetized by fentanyl and midazolam subcutaneous injection. Blood samples are collected from the saphenous vein, and the
blood collection was carried out before injection and after 10min, 30min, and 1, 2,
4, 6, and 24h after the injection of the formulation. Collected samples were then
diluted by heparin and then vortexed and then centrifuged. The uorescence of the
supernatant was measured by excitation at 535nm and emission at 620nm wavelength using a spectrophotometer (Böttger etal. 2016).
14.3.3.2 Radiolabeling
This is a very sensitive method for the determination of the pharmacokinetics of the
PEG moiety and the PEGylated compounds. In this method, a radioisotope is chemically included in the PEG or PEGylated molecules and then administered in the
small amounts. The radionucleotide atom keeps emitting the radiation which can be
then detected by gamma counter to monitor the emissions of the gamma rays and
the X-rays. The radioactive emissions can also be estimated by using the scintillation counter, which give an idea about α and β particle emissions. For this purpose,
many radioisotopes have been used by the researchers. In the case of PEGylated
liposomes and micelles, the radiolabeling is often useful for the pharmacokinetic
and biodistribution studies. The radioisotopes are generally attached to the surface
of a liposome or can be trapped inside the liposome. The main approaches for labeling of the liposomal surface:
(a) Anchoring of the metallic radioisotopes on the surface of the liposomes.
(b) Covalent linkage of the radiolabeled chelating agents to the surface of the
liposomes.
The pharmacokinetic estimation of the PEGylated peptides can be successfully
carried out by the incorporation of radioiodine, if the polypeptide backbone contains tyrosine residues (Cheng etal. 2012; Gaberc-Porekar etal. 2008).
14 Reversible PEGylation ofNanocarriers
409
14.4 Application ofReversible PEGylation
The PEGylation approach has been extensively employed for reducing the immunogenic properties, increasing enzymatic activities and most importantly, to prolong
clearance time (Kodera et al. 1998). The various applications of the reversible
PEGylation can be classied broadly as therapeutic and pharmaceutical applications.
(a) Therapeutic applications: Reversible PEGylation is being successfully
employed to modify the pharmacokinetic properties of immunological preparations, anticancer agents, and antibiotics. Recently, it was also found to be useful
in enzyme replacement therapy, RBC substitution, and some oxygen toxicity
diseases.
(b) Pharmaceutical applications: PEGylation aids the drug delivery, and the lipo-
somes are frequently PEGylated to impart the stealth properties. Proteins are
PEGylated for increasing their stability. The PEGylation approach also nds its
applications in targeted delivery of therapeutic agents.
14.4.1 Therapeutic Applications
Although the scientists have made the proteins having high compatibility with the
host immune system, these proteins often produce the negative immunogenic properties. So, PEGylated proteins can serve as the better means which do not affect the
immunity of the person. PEG stimulates the production of the antibodies, and it
itself does not induce the immunogenicity. PEG conjugation does not only cause of
the suppression of the hyperactive immune system, but also switches the immunogenic types of the proteins into the tolerogenic proteins, and hence, they are considered suitable for immunogenic protein administration. Severe attempts have been
made to screen such immunogenic proteins and have checked for their binding with
PEG moiety. The strategy for immunogenic protein PEGylation mainly involves:
(a) Identication of the conjugation site.
(b) Masking of the immunogenic sites of protein by PEG conjugates.
(c) Removal of the conjugate by immune system.
Then, conjugation sites are often indicated by the presence of the lysine and
serine, which, in due process of PEGylation, get substituted with cysteines. To
decrease the immunogenicity of the protein, the antigenic epitope of the protein is
masked with the hydrated PEG cloud. The improved immunological nature of this
protein is also depicted by the enhanced pharmacokinetic properties of the protein.
The half-life of the coated protein will also be prolonged because of the decrease
clearance of the proteins by immune system and also by glomerular ltration. After
its action, the conjugates and free PEG present in the circulation is removed by the
IgG and IgM antibodies (Shi etal. 2022).
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M. Mishra et al.
14.4.1.1 Anticancer Activity
Many enzymes possess the anticancer activity. Enzymes mainly cause depletion of
the nutrients required by the cancer cell growth, so the starvation of cancer cells is
caused without affecting the normal cells. Enzymes also face the similar issue like
other proteins including the short half-life inside the body and immunogenic nature.
An enzyme asparaginase is popularly used for the treatment of leukemia, but possess serious issue of causing immunogenic reactions which lead to the allergies and
anaphylactic shock. To combat the issues with asparaginase, PEGylated asparaginase was made by Enzon Inc. These PEGylated enzymes not only show decreased
immunogenicity, but also protect the enzymes from the degrading enzymes and also
prolong their circulation half-life (Higashi etal. 2020).
14.4.1.2 Antibiotic Administration
The antibiotics show limitations in use as anticancer drugs due to their low solubilities and side effects. The conjugates of antibiotics with PEG moiety can effectively
alter the solubility, permeability, and the distribution characteristics of the antibiotics in order to achieve the selective drug delivery with decreased side effects and
improved targeting at the desired sites of action. Scientists have successfully formulated the polymeric micelles of the antibiotics employing PEG moiety to overcome
the issues associated and achieve more patient-friendly therapy (Alavi etal. 2022).
14.4.1.3 Enzyme-Replacement Therapy
Sometimes, a disease state is associated with the alterations in the normal physiological levels of certain enzymes in the body. The enzyme activity maybe decreased
or completely lost, indicating a disease state. Enzon Inc. made mPEG-adenosine
deaminase, i.e., ADAGEN for the treatment of severe combined immunodeciency
disease (SCID). SCID causes partial or total dysfunction of immune system and
after receiving the formulation, patients show improved immune responses (Yadav
and Dewangan 2021).
14.4.1.4 Red Blood Cell Substitution
There are many diseases which are caused due to blood transfusions. To overcome
this issue, researchers have developed an alternative to natural blood for blood
transfusions. Articial blood can supply the increasing demand of blood and also
has increased half-life than the natural blood. The articial blood is generally comprised of the peruorocarbons, also known as “white bloods,” which resemble the
natural blood and are chemically related to the synthetic oils.
Red bloods are made by modication of the naked hemoglobin from RBCs, so as
to carry oxygen. Then major limitation associated with the articial blood is that the
hemoglobin present is not protected by the red blood cell membrane and hence
undergoes rapid clearance from the body and also leads to the formation of many
toxic metabolites. To combat all the limitations associated with the articial blood,
scientists have modied hemoglobin with PEG moiety of different molecular
weights, such as 1900, 4000, and 5000. The modication of hemoglobin with PEG
PEG
Aqueous core
14 Reversible PEGylation ofNanocarriers
411
moiety results in the increased half-life of the articial blood as well as decrease the
generation of the toxic metabolic products (Arkosi etal. 2023).
14.4.1.5 Oxygen Toxicity Diseases
The tissue damage is caused by the oxygen-free radicals. The various diseases
caused by radicals include inammation, thermal injury, and ischemia diseases.
Superoxidase dismutase is an antioxidant enzyme was supposed to avoid the oxidative damage caused by the radicals. Also, the catalase enzyme was also employed
for combating the harmful defects caused by the radicals. The disadvantage of
administering the enzymes such as superoxide dismutase or catalase is that these
enzymes possess very short half-life. When these enzymes are modied with the
PEG moiety, the modied enzymes shown increased half-life and also increased
uptake by the membrane. PEG moiety also shows the synergistic action with the
enzymes by inactivating the hydroxyl radicals invitro and also increase the function
of perfused kidney (Filpula and Zhao 2008).
14.4.2 Pharmaceutical Applications
14.4.2.1 PEGylated Liposomes
The PEGylated liposomes (Fig.14.7) show prolonged circulation of the liposomes
inside the body and also possess the clearance kinetics which is independent of the
dosage amount. The PEGylation hence improves the pharmacokinetic properties of
the drug substance and shows reduced toxicity to improve the therapeutic efciency
(Jain and Jain 2008).
By modication of the liposomal surface by PEG moiety, the efcacy of the liposome delivery can be improved by prolonging the circulation of the liposomes in the
blood. The liposomes conjugated with PEG are popularly known as “stealth liposomes” and possess increased half-life, decreased clearance from the body and
decreased volume of distribution. Also, the surface-modied liposomes are useful
in targeting particular organ for the delivery of the therapeutic agent. When the PEG
moiety is incorporated into the lipid bilayer of the liposome, it forms hydrated shell
Fig. 14.7 PEGylated liposome
Lipid bilayer
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M. Mishra et al.
that avoids the aggregation, hence improving the stability of the liposomal preparation. Also, inside the body, PEG helps to protect the liposomes from destruction by
plasma proteins and avoids the premature release of the liposomes inside the body.
PEGylation also avoids the interaction of the liposomes by the opsonin proteins and
avoids the liposomal uptake by RES (Thomas etal. 2022). PEGylation of the liposomes also serve as the effective means for passive targeting of the drug substance
to the desired action site. By virtue of the mechanism called enhanced permeation
and retention (EPR) effect, the liposomes can be preferentially accumulated in the
tumor areas and effectively deliver the drug there (Milla etal. 2012).
14.4.2.2 PEGylated Proteins
PEGylated proteins show improved stability and lowered immunogenicity, compared to unmodied protein (Fig.14.8). So, these modied proteins show sustained
action inside the body and reduce the frequency of administration. The modied
proteins are administered generally every week, but the unmodied proteins require
more frequent administration, i.e., once every 1 or 2days. The major limitation
associated with the PEGylation of the proteins is that it may cause the chemical
alteration of the protein molecule and sometimes shows the decreased therapeutic
response. To avoid this, the care should be taken to avoid such chemical interactions. The scientists have studied the effect of the PEGylation of the proteins for the
change in the pharmacokinetics, pharmacodynamics, and the biodistribution and to
achieve higher clinical efcacy (Harris and Chess 2003). Table14.2 contains the list
of some PEGylated proteins.
The largest group of proteins, which has been modied with the PEGylation is of
enzymes. Some enzymes, e.g., peroxidase, L-asparaginase, and alkaline phosphatase, upon conjugation with PEG moiety, showed excellent pharmacokinetic properties with increase stability (Milla etal. 2012). Some of the main enzymes which
are modied with PEGylation are given in Table14.3:
Fig. 14.8 PEGylated
protein
14 Reversible PEGylation ofNanocarriers
413
Table 14.2 PEGylated proteins
Table 14.3 PEGylated enzymes
PEGylated proteins Indication
Peglgrastim Neutropenia
Certolizumab pegol Rheumatoid arthritis
Pegloticase Chronic gout
CERA Anaemia
Pegademase Severe combined
immunodeciency disease
(SCID)
Pegaspargase Acute lymphoblastic leukemia
Peginterferon
Pegvisomant
Pegvisomant Acromegaly
Protein Application
Trypsin Increased proteolytic activity
Chymotrypsin Increased proteolytic activity
Ribonuclease Increased activity inside body
Tissue plasmin
activator
Interferons Antiviral applications
Interleukins Immune response
Elastase Increased hydrolyzing activity
Superoxide dismutase Antiinammatory activity
Alkaline phosphatase Increase enzyme activity
L-asparaginase Increased amidolytic activity
Peroxidase Increased enzyme activity invivo
Glucolactone oxidase Increase stability of the conjugate
Hepatitis C
Increase circulation half life
14.4.2.3 Targeted Delivery
When the nanoformulations are surface decorated with the PEG coatings, they specically bind the desired targeted receptors and increase the effectiveness of the
therapy, minimizing the drug loss by clearance and hence reduce the cost of the
therapy. Various researchers have successfully surface-modied the nanoformulations for targeting the receptors at brain, airways, nose, gastrointestinal tract, etc.
(Fig.14.9) (Jain and Jain 2008).
Reversible PEGylation forDelivery toAirways
The nanoformulation delivery to the airways is essentially required to treat many
ailments. However, the mucus layer present on the airways is rapidly cleared by the
mucociliary clearance mechanisms and hence possess signicant problems in the
drug delivery. The scientists have examined the penetration of the PEG-coated
nanoparticles through the sputum. Nanoparticles with 100nm size range are found
to effectively penetrate. Also, another barrier that hinders the drug action is the biolm. The PEG-modied nanoparticles can effectively pass through biolm. The
studies shown that when the nanoparticles sized about 100nm, they were unable to
414
PEGylated
PEGylate
d
liposome
PEGylated
liposome
Fig. 14.9 Reversible PEGylation for delivery to gastrointestinal tract, airways, brain and ocular
delivery
M. Mishra et al.
liposome
PEGylated
liposome
penetrate; however, when the 200nm nanoparticles well surface coated with the
PEG, they efciently penetrated the respiratory mucus (Wagner 2012).
Reversible PEGylation fortheDelivery toGastrointestinal Tract
There are many barriers in the gastrointestinal delivery of the drugs. The formulation should possess good stability properties in order to effectively overcome the
harsh environment. Researchers have studied the PEG coatings to improve the stability of the lipid based formulations. Various nonionic surfactants have been used
to improve the stability of the drug substance across the gastrointestinal tract. The
nanoparticles were further modied with the PEG coatings and found more effective than simple surfactant coatings. The nanoformulations also suffer because of
the rapid clearance of the mucus in the gastrointestinal tract. The PEG coatings
effectively increase the hydrophilicity of the lipid nanoparticles and hence minimize
the mucoadhesion. The PEG coatings also shown the decreased cytotoxicity. For
PEG coated nanoparticles who are targeting behavior, they show higher penetration
to the inamed tissue, but less penetration to the normal tissues (Sadekar and
Ghandehari 2012).
Reversible PEGylation fortheDelivery totheBrain
As described in the delivery to the airways, the PEGylation increases the nanoparticle delivery to the brain through nose. There are mainly two barriers for the drug
to reach the brain, rst is the blood-brain barrier (BBB) and the second is the tissue
extracellular matrix. Kannan group studied the penetration of the PEGylated
nanoparticles through extracellular matrix, and the nanoparticles coated with the
dense PEG coatings have been found to show the increased spread and the distribution, when studied in the rat model (Shechter etal. 2010).
14 Reversible PEGylation ofNanocarriers
415
Reversible PEGylation forOcular Delivery
The main hurdles associated with the ocular drug delivery are poor absorption and
the rapid clearance. Only less than 5% of the topically administered dose reaches
the ocular tissues. To overcome these issues, the nanoformulations should be
designed to possess the increased residence time and more penetration across the
tissues. The inert coating produced by the PEG forms the interactions between the
nanoparticle and the surface of an eye. PEG promotes mucoadhesion by formation
of the hydrogen bonds between the nanoparticle and the mucus. Sanders studied the
retinal drug delivery by using PEGylated nanoparticles and found that PEG coatings
improve the mobility and penetration through the vitreous chamber and hence
increase the drug delivery to the retina (Famili etal. 2014).
Reversible PEGylation fortheVaccine Delivery
Xua etal. have developed the hybrid nanoparticle-based nicotine vaccines by the
PEGylation approach and found that when the nanoparticles are coated with 20% or
lesser PEGylation, they possessed lesser stability. When the higher concentrations
of PEGylation were used, specically more than 30%, the nanoparticles shown the
nanoparticles with the compromised core-shell structure and also showed the slow
uptake by the dendritic cells. However, when the nanoparticles were coated by the
PEG concentrations in the range of 20–30%, they possessed higher stability and
enhanced uptake by the dendritic cells (Lv etal. 2022).
14.5 Challenges ofReversible PEGylation
Reversible PEGylation, a process involving the attachment of PEG chains to a molecule, has potential benets in drug delivery and biotechnology. However, it presents
several challenges, including controlled release, biocompatibility, stability, synthesis
and scalability, specicity, and immunogenicity (Veronese 2001). Controlled release
involves regulating the release of PEG chains to avoid premature release or insufcient stability. Biocompatibility concerns arise from the potential impact of PEG
breakdown products or detached chains on biological systems (Shechter etal. 2008).
Also, stability is crucial under various physiological conditions, such as pH, temperature, and enzyme presence. Developing efcient and scalable methods for synthesis is a signicant challenge, as it must be economically viable and suitable for
large-scale production. Specicity is another speed breaker in the path of PEGylation,
as non-specic interactions or unintended PEGylation may compromise the effectiveness of the reversible PEGylation strategy. Immunogenicity is essential to avoid
immune reactions that could impact therapeutic efcacy. Some of the key challenges
associated with Reversible PEGylation are as follows (Fig.14.10):
14.5.1 Design Complexity
Reversible PEGylation systems frequently entail the creation and synthesis of intricate compounds with the ability to release PEG under particular circumstances. It
416
Fig. 14.10 Key
challenges involved in
reversible PEGylation
M. Mishra et al.
can be difcult to make a balance between stability and controlled release; this calls
for close examination of the conjugate’s chemical makeup and physical
characteristics.
14.5.2 Kinetics ofRelease
To maximize the therapeutic impact, PEG release kinetics and rate must be regulated. PEG can be released gradually and under control without releasing too much
or too early because this is dependent on the particular stimuli or triggers used in the
reversible PEGylation system (Thakur etal. 2015).
14.5.3 Biological Environment Stability
In order for reversible PEGylation systems to release PEG, they must rst be stable
in the biological environment. Premature release into circulation and stability
against enzymatic degradation or other physiological circumstances are challenges
(Shechter etal. 2008).
14.5.4 Trigger Selection
Selecting the right trigger to release PEG is essential. Changes in pH, temperature,
or the presence of particular enzymes are examples of common triggers. However,
because it depends on the particular biological context and requirements, choosing
the best trigger for a certain application might be difcult (Zalipsky etal. 2007).