Synthesis of thiolated polymers is often more difficult with direct one-step methods (co-polymerization of monomers), and they are usually not employed for the synthesis of mucoadhesive polymers. As there is no theoretical obstacle, these methods are also briefly summarized. The biggest challenge is the high reactivity of thiol groups [139].
Radicals are able to cleave the hydrogen atom of the thiol yielding a thiyl radical.
Thiyl radical reacts with carbon-carbon double bonds in a thiol-ene reaction, thus, free a)
b)
radical polymerization of thiolated acrylates yield undefined products. Furthermore, thiols are used as chain transfer agents in free radical polymerization as thiyl radicals are able to start a new chain, and the molecular weight distribution can be controlled. Thiol groups also react through thiolate anions yielded by their deprotonation. The nucleophilic nature of thiolate anion may interfere anionic chain-growth polymerization as well as step-growth polymerization. The atmospheric oxidation of thiols to disulfides which also occurs with an anionic mechanism can also be problematic. The solution is either the utilization of a protecting group or the incorporation of the thiol groups after polymerization (indirect method).
A wide variety of protecting groups and corresponding deprotection reactions have been reported in the literature depending on the polymerization technique and the intended application. A possible strategy is to protect thiol groups as an unsymmetrical disulfide using a thiopyridyl compound as it was shown in the previous section. The protecting group can be removed by reduction of the disulfide, but it is more important that new functional groups can be introduced on the thiol groups by a thiol-disulfide exchange reaction also shown in the previous section. Ruffner and co-workers synthesized the copolymer of N-[(2-pyridyl)dithio-]ethyl methyacrylamide (PDTEMAAm) and N-(2-hydroxypropyl) methyacrylamide by free radical polymerization. The polymer was then reacted with a cysteine terminal peptide or a thiol terminal oligonucleodite in order to form conjugates that can be used in anti-cancer therapy [162].
Thiols can also be protected as thiolactones. Ring opening polymerization of a thiolactone can be initiated by a primary amine. Radical copolymerization of a styrene-thiolactone monomer with methyl methacrylate or styrene followed by the aminolysis of the pendant thiolactone leads to thiolated polymers as well. Thiolated poly(2-oxazolines) were prepared by living cationic ring opening polymerization using 4-methoxy-benzylsulfanyl moieties as protecting groups. Deprotection was performed with a mixture of trifluoroacetic acid and anisole. Other methods as well as the above mentioned ones have been recently reviewed by Le Neindre and Nicolay [139].
Enhancing solubility with cyclodextrin inclusion complexes
Short residence time of drug formulations is only one of the major challenges to overcome. The majority of new drug candidates are more lipophilic, have a higher molecular weight, and have a poor water solubility which leads to a low bioavailability.
About 40% of drugs with market approval and nearly 90% of molecules in the discovery pipeline are poorly water-soluble. [163] This is particularly problematic in the case of lipophilic ophthalmic drugs as the classic oil-based formulations (ointments) and suspensions are very uncomfortable to use and have a very low patient compliance.
According to the Biopharmaceutics Classification System (BCS) [164], poor solubility means that the highest dose of the drug cannot be dissolved in 250 ml water in the pH range of 1-7.5. There are several strategies to improve the solubility of these drugs including the utilization of prodrugs, pH modification, salt formation, using a co-solvent or a surfactant, forming solid dispersions, co-crystals or polymer micelles, amorphization, nanonization [163]. One of the most useful methods is the formation of cyclodextrin inclusion complexes. Cyclodextrins (CDs) are cyclic oligosaccharides
consisting of α-D-glucopyranose units that are united via α-1,4-linkage. Three major types of CDs are α, β and γ, consisting of 6, 7 and 8 glucopyranose units, respectively. CDs are toroidal cone shaped molecules due to the lack of free rotation of the bonds connecting the glucopyranose units. The cone has a predominantly hydrophobic central cavity and a hydrophilic outer surface, the hydrophobic cavity is able to entrap a drug molecule while the outer surface makes the complex soluble in water [165,166]. The relative size of CD to the guest molecule, the presence of key functional groups on the guest molecule, and thermodynamic interactions between CD, guest molecule and solvent are the key factors that enable the formation of an inclusion complex. Different derivatives of cyclodextrins were synthesized to optimize the solubility enhancing effect for different drugs.
Paclitaxel, for example, has a 4∙10-4 mM solubility in water that can be increased 13-fold by β-CD, and 99000-fold by 2,6-O-dimethyl-β-CD [165]. A few examples of marketed products include cefotiam hexetil or alprostadil complexed with α-CD, dexamethasone, nicotine with β-CD, 17β-estradiol with methylated-β-CD, diclofenac sodium with 2-hydroxypropyl-γ-CD [166]. CDs are also used for drug stabilization, drug protection from light, thermal and oxidative stress, taste masking of drugs, and reduced dermal, ocular or gastrointestinal irritation.
Higuchi and Connors [167] proposed phase-solubility diagram to describe the solubility profile and equilibrium constant of complex formation (Figure 1.13). In the case of Type A diagrams, the drug/CD complex is soluble in the aqueous complexation media. Positive (AP) and negative (AN) deviations from linearity (AL) may occur. Type B diagrams are observed when complex has limited solubility (BS) or insoluble (BI), therefore Type B profiles are not suitable for solubility enhancement of drugs.
Figure 1.13 Phase-solubility profiles. A: water soluble drug/CD complex. Profile may be linear (AL) or have a positive (AP) or negative (AN) deviation. B: drug/CD complexes with limited (BS) or no (BI) solubility. S0: solubility of the drug without cyclodextrin. [168]
One or more drug molecules can form a complex with one CD molecule and one or more CD molecules can form a complex with one drug molecule. However, if 1:1 stoichiometry is assumed (AL on Figure 1.13), equilibrium constant (Kc) can be calculated according to Eq. 1.14:
𝐾𝑐 = 𝑎 𝑆0(1 − 𝑎)
where S0 is the solubility of the drug in the complexation medium, and a is the slope of the phase-solubility diagram [167]. The optimal range of Kc for the solubilization of hydrophobic drug is 200-5000 1/M proposed by Szejtli [169] as in case of low values, solubilization is insufficient, while in case of higher values the absorption of the drug is limited.
The beneficial properties of cyclodextrins often utilized in mucoadhesive formulations either by simply mixing cyclodextrin in a formulation [170,171] or covalently attaching CD onto the chains of the mucoadhesive polymer [172,173]. As a results of covalent attachment, the stability of the complex can either increase or decrease.
Prabaharan et. al. [172] grafted chitosan on carboxymethyl chitosan (CMC) backbone using carbodiimide coupling, then further grafted with cysteine methyl ester hydrochloride. According to the authors, the mucoadhesive performance of the new polymer was 5-fold higher compared to CMC, and it provided slower release of the hydrophobic model drug, ketoprofen. Asim et. al. [174] described a microwave assisted method to prepare tetradeca-thiolated cyclodextrin (all primary OH groups at C-6 position and all secondary OH groups at C-2 position of β-CD was substituted by SH).
The authors reported increased viscosity upon addition to mucus, and increased retention time in flow through experiment indicating promising mucoadhesive performance.
Derivatives of poly(aspartic acid)
As we have seen in the earlier sections, several natural and synthetic polymers have been used to prepare hydrogels as drug delivery vehicles or other biomedical applications.
Natural polymers such as chitosan, hyaluronic acid, gelatine, gellan gum are appealing choices as they are biocompatible and biodegradable and they are able to mimic the properties of living tissues. However, they have several drawbacks limiting their widespread use. Batch-to-batch variability and wide molecular weight distribution impair reproducibility and they are less versatile as their functionalization requires catalysts and coupling agents, their chemical structure is difficult to be adjusted to a certain application.
Synthetic polymers, such as poly(acrylic acid) and its derivatives, on the other hand, can be synthesized in high chemical versatility with a precise control over molecular weight but their significant drawback is the lack of biodegradability.
Poly(aspartic acid) (PASP) and its derivatives are able to combine the favorable properties of natural and synthetic polymers and they can be a promising alternative base material of hydrogels in biomedicine as they are biocompatible [175–178], biodegradable [179–185] and they can be synthesized easily and with a remarkable versatility. PASP is a water-soluble, synthetic poly(amino acid) synthesized by the hydrolysis of polysuccinimide. Polysuccinimide is prepared by thermal polycondensation of aspartic acid (Figure 1.14A). The reaction is conducted at high temperature in a solvent mixture with high boiling point (mesitylene and sulfolane) or with no solvent. Phosphoric acid catalysis and reduced pressure facilitate the removal of water. The succinimide rings of the polymer are reactive structural units that are susceptible to various nucleophilic agents even under mild conditions. Alkaline hydrolysis of polysuccinimide yields the salts of poly(aspartic acid), polyaspartates. As the succinimide rings are asymmetrical, hydrolysis
(1.14)
yields a copolymer of α and β aspartic acid repeating units according to the reaction conditions and the type of the nucleophile. Due to the racemized chiral carbon in the structure, repeating units with both D and L configurations are present (Figure 1.14B) [186]. These structural features are rarely investigated, and often considered as reproducible for a given polymer derivative, hence, for the sake of simplicity, after Figure 1.14, only α aspartic acid repeating units with undetermined chirality will be displayed in this thesis. PASP has an inherent pH-responsive property due to the carboxylic group on the polymer backbone. The pKa values of PASP are 3.3 and 4.5 for α and β aspartic acid [6], respectively. Industrial uses of linear, unmodified poly(aspartic acid) is based on its chelating ability. It is used as an anti-scaling agent in cooling water systems or waste water treatment as it inhibits the deposition of Ca salts [186].
Figure 1.14 Synthesis route of different poly(aspartic acid) derivatives. (A) Precursor polymer, polysuccinimide is synthesized by the thermal polycondensation of L-aspartic acid. (B) Alkaline hydrolysis results in linear poly(aspartic acid). (C) Modified poly(aspartic acid) can be synthesized with carbodiimide chemistry, but (D) ring opening nucleophilic addition of primary amines offers a more convenient way. One or more modifying agents can be employed consecutively. (E) Subsequent hydrolysis results in modified poly(aspartic acid)s. (F) Polyaspartamides are obtained when all succinimide rings are reacted with a primary amine. (G) Polysuccinimide can be cross-linked with bifunctional primary amines. (H) Subsequent hydrolysis results in poly(aspartic acid) gels.
The chemical modification of poly(aspartic acid) is possible by e.g. carbodiimide mediated coupling but it is highly impractical considering the multiple reaction steps and an excess of coupling agents needed for the reaction (Figure 1.14C). It is significantly more convenient to react the precursor anhydride, polysuccinimide, with primary amines yielding poly(succinimide-stat-aspartamide) types of compounds (Figure 1.14D). These reactions take place under mild reaction conditions without any catalyst and usually with a high conversion. Depending on the chemical structure of the amines and the ratio of different repeating units in the copolymers, numerous functional polyaspartamides and modified poly(aspartic acid)s can be synthesized providing unparalleled versatility. Wide variety of PASP-based responsive polymers and hydrogels have been reported in the literature.
Partial opening of succinimide rings followed by hydrolysis yields modified poly(aspartic acid) derivatives (Figure 1.14E). The addition of monoamines results in alkyl-PASP derivatives with amphiphilic properties e.g. PASP with longer alkyl side chains (C16, C18) display micelle formation in aqueous medium [187,188]. Octylamine-grafted PASP can be used for the modification of tumor targeting liposomes. The hydrophobic alkyl chains “anchor” the liposomes while the polyelectrolyte backbone stabilizes them at pH = 7.4 and retards drug release[189]. The solubility and pKa of PASP can be adjusted by the type and the grafting ratio of alkyl side-groups. Increasing grafting ratio and length of alkyl groups decreases solubility and shifts pKa to higher values.
Considering the biodegradability of PASP, these derivatives can be useful for the preparation of enteric coatings as an alternative to non-degradable acrylic materials [176].
PASP with thiol pendant groups was prepared by reacting PSI with cysteamine. Cross-linking by aerial oxidation results in a PSI gel, and hydrolysis and subsequent reduction yield the water-soluble thiolated PASP. The polymer shows redox-induced reversible sol-to-gel transition in aqueous environment due to reversible thiol-disulfide transition [190].
Thiolated PASP nanogels prepared in water-in-oil miniemulsion degrade in reductive environment which can be exploited in targeted drug delivery [191]. Potential of thiolated PASP as a second generation mucoadhesive polymer was assessed in our group. It shows good adhesion strength on mucin due to its in situ gelling but derivatives with different thiol content were not investigated [192]. Swellable hydrogel matrix of thiolated PASP was prepared by reactive electrospinning. Due to its unique morphology, this matrix is promising as artificial extracellular matrix material [193].
Succinimide rings of PSI can be reacted completely with one or, in consecutive way, more primary amines to obtain polyaspartamide homo- and copolymers (Figure 1.14E, F). Physical properties of these derivatives – glass transition temperature, solubility, gelation temperature – can be adjusted by the ratio of different repeating units.
Amphiphilic polyaspartamides with dodecyl and hydroxyalkyl side chains show temperature dependent sol-to-gel transition potentially applicable as in situ gelling injectable system [194]. Cationic thermogelling derivatives with N,N-diisopropylaminoethyl and dodecyl side-chains were found to be useful for chlorambucil release. Hydroxyl group of a polyaspartamide derivative with N,N-diisopropylaminoethyl and hydroxyethyl side chains can be cross-linked with diisocyanates yielding a pH and thermoresponsive hydrogel [182,195]. Polyaspartamides with a combination of alkylamino side chains and alkyl side chains are promising candidates as film forming materials for taste masking tablet coatings. These polymers have adjustable glass transition temperature and dissolution profile with slow dissolution rate at the pH of the
mouth and quick dissolution rate at the pH of the stomach [178]. Electrospun matrices prepared of cationic PASP-derivatives have a prompt dissolution due to the nanosized electrospun fibers which can be utilized in sublingual drug delivery [196]. Similar cationic derivatives can be used as gene-carriers as they form polyionic complexes (polyplexes) with negatively charged DNA and RNA molecules and effective cell transfection was achieved [197]. Poly{N′-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}
(Pasp(DET)) has also high transfection efficiency, moreover, as it is able to degrade under in vivo circumstances in a few days, its accumulation in the cells is prevented. It was found that the degradation is a self-catalytic reaction between the backbone and the side-chain amide nitrogen. Possible application of Pasp(DET) in the treatment of spinal cord injury was demonstrated [181,198,199]. Transfection of small interfering RNA (siRNA) is challenging due to its low molecular weight. Thiol-modified siRNA can be covalently attached to Pasp(DET) with 2-(2-pyridinyldithio)ethyl side groups by thiol-disulfide exchange improving the stability of the polyplex and allowing efficient siRNA release under reductive conditions in the cytoplasm [200].
The reaction of PSI and bifunctional or multifunctional amines in DMF or DMSO results in chemically cross-linked networks (Figure 1.14G). The PASP gels obtained by their hydrolysis (Figure 1.14H) have a pH-responsive swelling due to the polycarboxylic acid character. At acidic pH, the carboxyl groups of PASP are protonated, the swelling degree of the gel is low. Increasing the pH results in the gradual deprotonation of carboxyl groups accompanied by the abrupt increase of the degree of swelling due to the better solvation of negatively charged carboxylate anions [6]. PASP-based hydrogels are usually developed for cell culturing/tissue engineering applications. Supermacroporous PASP hydrogel linked with 1,4-diaminobutane is prepared by conducting the cross-linking reaction under the freezing point of DMSO [201]. The large pores and open pore structure of the gel is ideal for the accommodation of the living cells. Redox and pH responsive PASP gels are synthesized by employing a redox-inert and a redox-sensitive cross-linker. The gel swells reversibly in response to reduction and oxidation as the redox-sensitive cross-links break and reform but does not dissolve due to the redox-inert cross-links. The gel can be synthesized in a one-step method by cross-linking PSI with 1,4-diaminobutane and cystamine [202] or in a consecutive method, where PSI is modified with cysteamine first, then in the second step, the polymer is cross-linked with 1,4-diaminobutane [203]. These gels can support the survival, proliferation, as well as the migration of human osteoblast cells. It was shown that the significant increase of cell viablility is due to the presence of thiol groups in the gel [179].
Scope
The unique properties of polymer hydrogels make them excellent candidates for a wide range of biomedical uses such as in situ gelling and responsive drug delivery systems. Biocompatibility, biodegradability and chemical versatility of poly(aspartic acid) have driven us to choose it as a base polymer in this research. We aimed to prepare poly(aspartic acid) derivatives and hydrogels with different properties depending on the targeted application. Materials and experimental methods that were used throughout the entire research are summarized in Chapter 2, specific methods are described at the beginning of each further chapter.
Drug absorption through mucous membranes can be improved by mucoadhesive drug delivery vehicles. These dosage forms provide a longer residence time and a more efficient absorption at the site of action. In particular, semi-solid and liquid mucoadhesive dosage forms can improve the low bioavailability of ophthalmic drugs. The mucoadhesive properties of the base materials of such dosage forms is enhanced by attaching thiol pendant groups onto the polymer backbone to provide covalent bonds between the dosage form and the cysteine-rich subdomains of mucin protein building up the mucus layer. Although the semi-solid and liquid ophthalmic formulations have the potential to form strong interactions with the mucus layer, the lack of cohesive properties in the dosage forms prevent them to achieve sufficiently long residence time. The answer to this challenge might be an in situ gelling polymer solution which can be chemically cross-linked through disulfide bonds. The solution of the hydrophilic polymer has a relatively low viscosity allowing easy application and good spreadability on the mucosal surface and effective interpenetration of the polymer and the mucin chains at the interface. Chemical net-points forming during the in situ gelling process cross-link the interpenetrating network of the mucin and the polymer and also ensures the cohesive structure of the hydrogel. Accordingly, one of the aims of this research work was to prepare a poly(aspartic acid) derivative that is able to fulfil these requirements. In Chapter 3, a novel synthesis method is reported to prepare thiolated poly(aspartic acid) with controllable and large thiol content. Oxidation induced in situ gelation was monitored with oscillation rheology and results on mucoadhesive properties are also reported. Sustained release potential of the gels is demonstrated with an in vitro drug release experiment using a water-soluble ophthalmic drug. The effect of the thiol content of the polymers was evaluated in each experiment.
Poorly soluble ophthalmic drugs such as prednisolone are commonly administered as oily formulations or aqueous suspensions resulting in low bioavailability and patient compliance. The problem can be addressed by improving water solubility through cyclodextrin inclusion complexes. The possibility to combine the advantages of cyclodextrin complexes and mucoadhesive delivery is demonstrated in Chapter 4. The thiolated poly(aspartic acid) was further modified with β-cyclodextrin, the synthesis method and the analysis of the chemical structure is described. The properties of the inclusion complexes formed by prednisolone and the cyclodextrin-modified polymer were analyzed with a phase-solubility study and X-ray powder diffraction. The effect of the covalently attached cyclodextrin on the drug release was also evaluated.
The results gained in Chapter 3 and 4 encouraged us to take a step further and aim to improve the properties of thiolated poly(aspartic acid) by preactivating the polymer via
attaching thiopyridyl type subunits onto the thiol pendant groups. These S-protected moieties are able to form disulfide bonds with the free thiols in a facile thiol-disulfide exchange reaction without the addition of an oxidant. In Chapter 5, the synthesis method and the chemical characterization of the S-protected thiolated poly(aspartic acid) is reported. In situ gelling properties and interaction with mucin were investigated. The properties of in situ gels were thoroughly examined in terms of stability, stiffness and drug release. In vitro cytotoxicity experiments were carried out to assess the biocompatibility of the polymers.
Apart from the covalent disulfide bonds, mucoadhesive properties are largely defined by the secondary interactions between the mucin protein and the mucoadhesive polymer.
Electrostatic interactions depending on the charge of the polymer backbone are particularly crucial. In Chapter 6, three cationic polyaspartamides were synthesized and their interactions with the colloidal dispersion of porcine gastric mucin were studied in comparison with those of poly(aspartic acid) and chitosan. A series of physicochemical methods was utilized in order to gain better understanding on interactions including turbidimetry, dynamic light scattering and zeta potential measurement. These findings can contribute to a better design of mucoadhesive polymers in the future such as combining the advantage of the cationic character with that of the thiolation or in situ cross-linking.
The reversed process of in situ gelation, stimuli-induced degradation of a hydrogel holds interesting possibilities as well. Enzymes are particularly promising stimuli due to the substrate specificity which can be exploited when designing tissue engineering scaffolds or drug delivery vehicles. In Chapter 7, the synthesis of a trypsin-responsive poly(aspartic acid) hydrogel is described. The hydrogel utilizes a tetrapeptide cross-linker with a trypsin-specific cleavage site. Degradation by trypsin, enzyme-controlled drug release and preliminary biocompatibility tests are included in the chapter. In the future, the synthetic method can be extended to design degradable hydrogels that are responsive to any protease enzyme.
In the final chapter of this thesis, in Chapter 8, the most important results of this research work are briefly summarized, and the thesis points of the work are listed. The experimental results obtained during this work led to several interesting findings, which will hopefully contribute to the future biomedical application of poly(aspartic acid) based materials.
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