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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5640_Библиотеки_им_академика_М_И_Перельмана.pdf
Drug Delivery in Rare Diseases: Orphan Drugs and Therapies 419
Table 3
Drugs carried by poly(lactic-co-glycolic acid, PLGA) nanoparticles
Drug Delivery Disease Preparation method
Docetaxel Enhanced solubility and tumor
targeting
Paclitaxel Improved therapeutic index and
reduced side effects
Curcumin Enhanced bioavailability and
targeted delivery
Rifampicin Sustained release for tuberculosis
treatment
Acyclovir Topical delivery for antiviral
treatment
siRNA (small
interfering RNA)
Insulin Controlled release for diabetes
Donepezil Improved delivery for Alzheimer’s
4.2 Methods
4.2.1 Reagents
Gene silencing for specific targets Cancer Electrospraying
management
disease
1. PLGA (50:50 or desired ratio)
2. Polyvinyl alcohol (PVA) (stabilizer)
3. Dichloromethane (DCM) or acetone (solvent)
4. Drug to be encapsulated
5. Deionized water
6. Magnetic stirrer
7. Ultrasonicator
8. Centrifuge
9. Rotary evaporator
10. Dialysis membrane
11. Lyophilizer
Non-small cell lung
cancer
Ovarian cancer Nanoprecipitation
Inflammatory
bowel disease
Tuberculosis Spray drying
Herpes simplex
virus infection
Type 1 diabetes Solvent displacement
Alzheimer’s disease Nanoprecipitation-
Single emulsion solvent
evaporation
Double emulsion
solvent evaporation
Emulsion-solvent
evaporation
ultrasonication
4.2.2 Procedure 1. Dissolve PLGA in DCM or acetone at a concentration of
10–20 mg/mL. The choice of solvent depends on the solubility of the drug and PLGA [60].
2. Dissolve or disperse the drug in the PLGA solution. Ensure
that the drug is evenly distributed within the solution.
3. Add the
PLGA-drug solution dropwise to an aqueous PVA
solution (1–2% w/v) under vigorous stirring to form an oilin-water emulsion. The typical PLGA to PVA weight ratio
ranges from 1:10 to 1:20.
420 Anil Kumar et al.
4. Sonicate the emulsion using an ultrasonicator for 2–5 min to
5. Transfer the emulsion to a rotary evaporator and evaporate the
6. Centrifuge the nanoparticle suspension at 10,000 rpm for
7. Dialyze the nanoparticle suspension against deionized water
8. Freeze-dry the purified nanoparticle suspension to obtain a dry
5 Polycaprolactone (PCL)
reduce the size of the droplets and achieve a uniform distribution of nanoparticles.
solvent under reduced pressure at room temperature until the
nanoparticles are formed. This step ensures the removal of the
organic solvent, leaving behind PLGA nanoparticles suspended
in the aqueous phase.
20 min to pellet the nanoparticles. Wash the pellet with deionized water to remove excess PVA and unencapsulated drug.
for 24 h using a dialysis membrane to remove any residual
solvent and small molecules.
nanoparticle powder. Store the powder at -20 °C for
future use.
5.1 Surface
Modification
Woodruff and Hutmacher [65] highlighted the versatility of polycaprolactone in drug delivery systems. PCL’s slow degradation rate
makes it suitable for long-term drug release applications, which is
beneficial in treating chronic conditions associated with rare dis-
65]. PCL nanoparticles can be prepared using several tech-
eases [
niques, with nanoprecipitation and emulsification-solvent
evaporation being the most common. This method involves dissolving PCL in a water-miscible organic solvent such as acetone. The
organic solution is then added drop by drop to an aqueous phase
containing a stabilizer like polyvinyl alcohol (PVA) under constant
stirring. The rapid diffusion of the organic solvent into the aqueous
phase leads to the formation of PCL nanoparticles [
66]. In another
method, PCL is dissolved in a volatile organic solvent (e.g., dichloromethane) and then emulsified in an aqueous phase containing a
surfactant like PVA. The organic solvent is evaporated under
reduced pressure or ambient conditions, resulting in the formation
of PCL nanoparticles. This method is particularly useful for the
encapsulation of hydrophobic drugs [
67].
Surface modification of PCL nanoparticles is crucial for improving
their biocompatibility, targeting capability, and circulation time.
Common techniques include polyethylene glycol coating, which
enhances the hydrophilicity of nanoparticles and reduces opsonization, thus prolonging their circulation time in the bloodstream
68]. Ligand conjugation with specific antibodies or peptides
[
improves the efficiency of drug delivery [
69].
Drug Delivery in Rare Diseases: Orphan Drugs and Therapies 421
5.2 pH Sensitivity and Stability
PCL nanoparticles exhibit pH sensitivity, which can be exploited
for controlled drug release. The stability of PCL nanoparticles is
influenced by the pH of the environment. In acidic conditions, the
degradation of PCL is accelerated, leading to a faster release of the
encapsulated drugs. This property is advantageous for targeting
acidic environments such as tumor tissues or intracellular compartments [
70]. At neutral or basic pH, PCL nanoparticles are more
stable and degrade slowly, providing a sustained release of the drug
[65]. The preparation of PCL nanoparticles is generally performed
at neutral pH to ensure stability and effective encapsulation. Maintaining a neutral pH during preparation ensures the stability of
both the polymer and the drug, leading to uniform nanoparticle
5.3 Methods
5.3.1 Materials
formation and high encapsulation efficiency [
1. ε-Caprolactone
2. Stannous Octoate, Sn(Oct)
2
71].
3. Methanol
4. Dimethyl sulfoxide (DMSO) or chloroform
5.3.2 Procedure (Fig. 3) 1. Add a predetermined amount of ε-caprolactone (CL) into a
round-bottom flask.
Fig. 3 Preparation of chitosan-based nanoparticles
422 Anil Kumar et al.
2. Introduce stannous octoate (Sn(Oct)2) as a catalyst in a 1:2000
molar ratio of catalyst to monomer.
3. Purge the reaction mixture with nitrogen gas for 10 min to
remove any oxygen.
4. Heat the reaction mixture to 110 °C under a nitrogen atmosphere with constant stirring.
5. Maintain the temperature and stirring for 24 h to complete the
polymerization process.
6. Cool the mixture to room temperature after the polymerization is complete.
7. Dissolve the polymerized product in DMSO or chloroform.
8. Precipitate the polymer by adding the solution drop by drop
into cold methanol with vigorous stirring.
9. Collect the precipitated polymer by filtration and wash with
methanol to remove unreacted monomers and catalyst
residues.
10. Dry the collected polymer in a vacuum oven at 40 °C for 24 h
to remove any residual solvents.
5.4 Drug Loading
1. Dissolve the purified PCL in DMSO or another suitable
solvent.
2. Add the drug intended for delivery into the PCL solution,
ensuring complete dissolution.
3. Mix the solution thoroughly to achieve uniform drug
distribution.
6 Chitosan-Based Systems
Chitosan is derived from chitin, a natural polysaccharide found in
the exoskeletons of crustaceans and insects, as well as in the cell
walls of fungi [
ity, and non-toxicity, it is widely used in various biomedical applications, including drug delivery systems, wound dressings, and tissue
engineering [
network that absorbs and retains significant amounts of water,
making it suitable for applications requiring moisture retention
and controlled release of substances [
nanoparticles have been used for the delivery of nucleic acids and
proteins in treating genetic and metabolic disorders [
the most common methods for preparing chitosan nanoparticles is
ionic gelation, involving the interaction between positively charged
chitosan and a negatively charged polyanion like sodium tripolyphosphate (TPP) [
72, 73]. Due to its biocompatibility, biodegradabil-
74, 75]. When used as a hydrogel, chitosan can form a
76, 77].
Chitosan-based
78]. One of
78].
Drug Delivery in Rare Diseases: Orphan Drugs and Therapies 423
6.1 Encapsulation of Nucleic Acids and Proteins
6.2 Surface
Modification
6.3 pH Sensitivity and Stability of Chitosan Nanoparticles
Chitosan can form complexes with nucleic acids due to its positive
charge, which facilitates binding to the negatively charged nucleic
acids. This electrostatic interaction helps in the efficient encapsulation and protection of nucleic acids [
79]. Proteins can be
encapsulated within chitosan nanoparticles through adsorption,
covalent bonding, or entrapment during the nanoparticle formation process. The mild conditions used in the preparation process
help maintain the structural integrity and activity of the
proteins [
80].
The surface of chitosan nanoparticles can be modified with targeting ligands such as folic acid, antibodies, or peptides to enhance the
specificity of drug delivery to target cells or tissues [81]. Similar to
liposomal formulations, chitosan nanoparticles can be pegylated to
improve their circulation time and reduce immune
recognition [
82].
Chitosan-based nanoparticles are highly sensitive to pH changes,
which can significantly impact their stability, encapsulation efficiency, and drug release profiles. Chitosan is a weak base with a
pKa value around 6.5, which means it is more soluble in acidic
conditions where it is protonated. The preparation of chitosan
nanoparticles typically occurs at a pH range of 4.0–5.5 to ensure
that chitosan remains in a dissolved and protonated state, facilitating efficient nanoparticle formation through ionic gelation
78, 83]. These nanoparticles are often maintained at a slightly
[
acidic pH, usually around 5.0–6.0, to maintain their protonation
and prevent aggregation, ensuring stability over time [84].
6.4 Methodology
6.4.1 Reagents
1. Chitosan (medium molecular weight)
2. Acetic acid (1% v/v)
3. Sodium tripolyphosphate (TPP)
4. Drug to be encapsulated
5. Deionized water
6. Magnetic stirrer
7. Ultrasonicator
8. Centrifuge
9. pH meter
10. Dialysis membrane
11. Lyophilizer
424 Anil Kumar et al.
6.4.2 Procedure
The entire procedure of chitosan nanoparticles is summarized as
follows (Fig.
3):
1. Dissolve chitosan in 1% (v/v) acetic acid solution to obtain a
1–2 mg/mL chitosan solution [
85].
2. Stir the solution overnight at room temperature using a magnetic stirrer until the chitosan is completely dissolved.
3. Prepare a TPP solution in deionized water at a concentration of
1 mg/mL [
86].
4. Dissolve the drug in the chitosan solution under magnetic
stirring. The concentration of the drug depends on the desired
drug loading efficiency [87].
5. Add the TPP solution drop by drop to the chitosan-drug solution under continuous stirring. The typical chitosan to TPP
weight ratio ranges from 3:1 to 5:1 [88]. This leads to the
ionic gelation of chitosan and the formation of nanoparticles.
6. Sonicate the mixture using an ultrasonicator for 5–10 min to
reduce the size of the nanoparticles and achieve a uniform
distribution [89].
7. Adjust the pH of the nanoparticle suspension to 5.5 using 1 M
NaOH or HCl, as chitosan nanoparticles are more stable at this
pH [90].
8. Centrifuge the suspension at 10,000 rpm for 20 min to separate the nanoparticles. Wash the pellet with deionized water to
remove excess TPP and unencapsulated drug [91].
9. Dialyze the nanoparticle suspension against deionized water
for 24 h using a dialysis membrane to remove residual acetic
acid and other small molecules [92].
10. Freeze-dry the purified nanoparticle suspension to obtain a dry
nanoparticle powder. Store the powder at -20 °C for future
use [93].
7 Dendrimers
Dendrimers are highly branched, synthetic macromolecules with a
tree-like structure, known for their unique and intricate architecture. These macromolecules are synthesized through a controlled
step-by-step process, resulting in a symmetrical and highly
branched structure that resembles a tree with multiple branches
extending from a central core [
94, 95]. For instance, polyamidoa-
mine (PAMAM) dendrimers are synthesized starting from an ethylenediamine core, with each generation involving the addition of
methacrylate units, resulting in a highly branched architecture
[
The branching occurs in layers, or generations, where each
96].
successive generation adds new branches, increasing the overall size
and complexity of the dendrimer [
96].
Drug Delivery in Rare Diseases: Orphan Drugs and Therapies 425
Fig. 4 Dendrimer
PAMAM dendrimers can be synthesized up to the tenth generation, with each generation doubling the number of terminal
groups and significantly increasing the molecular weight and size
97]. Their well-defined three-dimensional structure provides
[
numerous advantages for drug delivery purposes. The highly
branched architecture creates internal cavities within the dendrimer, which can encapsulate a variety of guest molecules, including
drugs, within these cavities (Fig.
4) [98, 99]. T
his encapsulation
capability is particularly beneficial for enhancing the solubility and
stability of hydrophobic drugs, which are often challenging to
deliver effectively in their native form [
Dendrimers can
also be functionalized with targeting ligands,
100] (Table 4).
such as antibodies or peptides, allowing for targeted delivery and
controlled release of therapeutic agents [101, 102]. For instance,
426 Anil Kumar et al.
Table 4
Dendrimer-based drugs
Drug Description Disease
Preparation
method
Doxorubicin Enhanced tumor penetration and reduced
cardiotoxicity
Camptothecin Improved water solubility and targeted
delivery
Curcumin Increased bioavailability and targeted
delivery to inflammatory sites
Genevec
(siRNA)
5-fluorouracil
(5-FU)
Ibuprofen Localized
Insulin Controlled release for
Acyclovir Topical deliver
Gene silencing for specific targets Cancer Michael addition
Controlled release for colon cancer treatment Colon cancer Co-precipitation
delivery for pain management
diabetes management
y for antiviral
treatment
dendrimers functionalized with folic acid have been used to target
cancer cells overexpressing folate receptors, thereby increasing the
specificity of drug delivery [103]. The surface of dendrimers can
also be modified to release their payload in response to specific
stimuli, such as pH changes, temperature variations, or enzymatic
activity [
96]. This controlled release mechanism ensures that the
drug is released at the desired site of action and at the optimal
therapeutic concentration. For example, dendrimers designed to
degrade in acidic environments can release their payload in the
acidic tumor microenvironment, enhancing the targeted delivery
of anticancer drugs [
98].
In addition to drug delivery, dendrimers have been explored for
a range of other biomedical applications. Their ability to form
stable complexes with nucleic acids, such as DNA and RNA,
makes them promising candidates for gene delivery and gene therapy [
97]. For instance, they have been used to deliver small inter-
fering RNA (siRNA) for the treatment of genetic disorders,
improving the stability and cellular uptake of siRNA
molecules [
104].
Cancer Divergent
synthesis
Cancer Convergent
synthesis
Inflammatory
bowel disease
Ar
thritis
Type 1
Herpes simplex
diabetes
virus infection
Click chemistry
reaction
Encapsulation
Post-generation
modification
Grafting
7.1 Antisense Oligonucleotides
Antisense oligonucleotides (ASOs) are short, synthetic strands of
DNA or RNA that can bind to specific mRNA molecules, blocking
their ability to produce proteins. This mechanism makes ASOs
effective in treating genetic disorders by silencing disease-causing
genes. Dendrimer nanoparticles have been employed to deliver
Drug Delivery in Rare Diseases: Orphan Drugs and Therapies 427
ASOs for conditions such as Duchenne muscular dystrophy
(DMD), a severe muscle-wasting disease [
105, 106]. They enhance
the delivery of ASOs by improving their stability and cellular
uptake. For example, PAMAM dendrimers conjugated with ASOs
targeting dystrophin mRNA have shown increased efficiency in
muscle cells, offering potential therapeutic benefits for DMD
patients [
107, 108]. The ability to modify the surface of dendrimers
with targeting ligands allows for specific delivery to muscle tissues,
reducing off-target effects and enhancing therapeutic
outcomes [
98].
7.2 Small-Interfering RNA (siRNA)
7.3 Chemotherapeutic Agents
They are ideal carriers for siRNA due to their ability to form stable
complexes with these nucleic acids, protecting them from degradation and facilitating cellular uptake. For instance, dendrimer-based
siRNA delivery systems have been developed to target and silence
the transthyretin gene, demonstrating significant gene silencing
and therapeutic effects in preclinical models [
100, 104]. The sur-
face functionalization of dendrimers with polyethylene glycol
(PEG) or other stabilizing agents further enhances the biocompatibility and circulation time of siRNA, improving its therapeutic
efficacy [
Dendrimer-based delivery systems
109].
have been extensively investigated for the delivery of chemotherapeutic agents, particularly for
treating rare cancers such as glioblastoma. Glioblastoma is an
aggressive brain tumor with poor prognosis and limited treatment
options. The unique structure of dendrimers allows for the encapsulation and targeted delivery of chemotherapeutic drugs, improving their solubility, stability, and therapeutic index [
110, 111]. For
example, dendrimers have been used to deliver drugs like doxorubicin and paclitaxel to glioblastoma cells. These dendrimer-drug
complexes can cross the blood-brain barrier and release the drug
directly at the tumor site, enhancing its cytotoxic effects on cancer
cells while minimizing damage to healthy tissues [
97, 112]. Further-
more, the surface modification of dendrimers with targeting
ligands, such as transferrin or folic acid, facilitates the selective
targeting of glioblastoma cells, increasing the specificity and efficacy
of the treatment [
113, 114].
7.4 Methods of
Preparation of
Dendrimer-Based
Nano
particles
7.4.1 Divergent Method The divergent method, also known as the “core-outward” method,
Two methods are used to prepare dendrimer based nanoparticles.
begins with a core molecule and builds outward in a stepwise
fashion [95, 115]. Start with a multifunctional core molecule,
428 Anil Kumar et al.
such as ethylenediamine for polyamidoamine (PAMAM) dendrimers [
116, 117]. Dissolve the core molecule in a suitable solvent,
such as methanol or dimethyl sulfoxide [
117, 118]. Add an excess
of acrylate or amine-based monomers, such as methyl acrylate for
PAMAM dendrimers [100, 119]. Stir the reaction mixture at room
temperature or slightly elevated temperatures, typically between
25 and 50 °C, for a specific period, usually 24–48 h, to ensure
complete reaction [
104, 116]. Purify the resulting product
using
techniques such as precipitation, dialysis, or ultrafiltration to
remove unreacted monomers and by-products [
98, 118].
To synthesize the second generation (G2), activate the ter minal
groups of the first-generation dendrimer by adding a cross-linking
agent such as ethylenediamine [112, 119]. Repeat the monomer
addition step using the same or different monomers to form the
second-generation dendrimer [
97, 114]. Maintain similar reaction
conditions as for the first generation, then purify the secondgeneration dendrimer to remove unreacted reagents and
by-products [
113, 115]. Continue this process for subsequent
generations, repeating the monomer addition, activation, and purification steps for each successive generation until the desired generation, such as G5 or G10, is achieved [
95, 116]. Optimize
reaction times, temperatures, and purification methods to ensure
high purity and yield at each generation [104, 116].
Finally, conduct a thorough purification of the final dendrimer
product using advanced techniques such as high-perfor mance liquid chromatography (HPLC) or size-exclusion chromatography
(SEC) [
98, 118]. Characterize the final dendrimer using spectro-
scopic methods such as nuclear magnetic resonance (NMR) and
mass spectrometry (MS), along with chromatographic techniques,
to confirm its structure, molecular weight, and purity [
111, 112].
7.4.2 Convergent Method This method, also known as the “outside-inward” method, starts
with the synthesis of dendritic branches, which are then attached to
a core molecule [115, 116]. Select suitable monomers for the
dendritic branches, such as amine-based monomers
[
100, 104]. Begin by synthesizing the outermost layer of the
dendrimer, typically the third or fourth generation [
118, 120]. Dis-
solve the monomers in a suitable solvent, such as methanol or
DMSO, and carry out the polymerization reaction at controlled
temperatures, generally between 25 and 50 °C, with continuous
stirring [
114, 117]. P
urify the resulting branched structure using
precipitation, dialysis, or ultrafiltration to remove unreacted monomers and by-products [
Next, activate
113, 119].
the terminal groups of the outer branches and
couple them with intermediate-generation branches
114, 118]. Maintain the reaction mixture at appropriate tempera-
[
tures, typically 25–50 °C, with continuous stirring [
98, 112]. Purify