Reduction-responsive polymers for drug delivery in cancer therapy—Is there anything new to discover?

© 2020 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals LLC. Among various types of stimuli-responsive drug delivery systems, reduction-responsive polymers have attracted great interest. In general, these systems have high stability in systemic circulation, however, they can respond quickly to differences in the concentrations of reducing species in specific physiological sites associated with a pathology. This is a particularly relevant strategy to target diseases in which hypoxic regions are present, as polymers which are sensitive to in-situ expressed antioxidant species can, through a local response, release a therapeutic at high concentration in the targeted site, and thus, improve the selectivity and efficacy of the treatment. At the same time, such reduction-responsive materials can also decrease the toxicity and side effects of certain drugs. To date, polymers containing disulfide linkages are the most investigated of the class of reduction-responsive nanocarriers, however, other groups such as selenide and diselenide have also been used for the same purpose. In this review article, we discussed the rationale behind the development of reduction-responsive polymers as drug delivery systems and highlight examples of recent progress. We include the most popular design methods to generate reduction-responsive polymeric carriers and their applications in cancer therapy, and question what areas may still need to be explored in a field with already a very large number of research articles. Finally, we consider the main challenges associated with the clinical translation of these nanocarriers and the future perspectives in this area. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies

Versatile routes to functional RAFT chain transfer agents through the Passerini multicomponent reaction

The widespread adoption of RAFT polymerization stems partly from the ease and utility of installing a functional chain transfer agent onto the ends of the generated polymer chains. In parallel, the Passerini multicomponent reaction offers great versatility in converting a wide range of easily accessible building blocks to functional materials. In this work, we have combined the two approaches such that a single, commonly available, RAFT agent is used in Passerini reactions to generate a variety of multifunctional RAFT chain transfer agents containing ester linkages. Reactions to generate the multifunctional RAFT agents took place under mild conditions and in good yields. The resulting Passerini-RAFT agents were able to exert control over radical polymerization to generate materials of well-defined molecular weights and dispersity. Furthermore, the presence in these polymer cores of ester and amide functionality through the Passerini chemistries, provided regions in the materials which are inherently biodegradable, facilitating any subsequent biomedical applications. The work overall thus demonstrates a versatile and facile synthetic route to multi functional RAFT chain transfer agents and biodegradable polymers

Dual Bioresponsive Antibiotic and Quorum Sensing Inhibitor Combination Nanoparticles for Treatment of Pseudomonas aeruginosa Biofilms In Vitro and Ex Vivo

Many debilitating infections result from persistent microbial biofilms that do not respond to conventional antibiotic regimens. A potential method to treat such chronic infections is to combine agents which interfere with bacterial biofilm development together with an antibiotic in a single formulation. Here, we explore the use of a new bioresponsive polymer formulation derived from specifically modified alginate nanoparticles (NPs) in order to deliver ciprofloxacin (CIP) in combination with the quorum sensing inhibitor (QSI) 3-amino-7-chloro-2-nonylquinazolin-4(3H)-one (ACNQ) to mature Pseudomonas aeruginosa biofilms. The alginate NPs were engineered to incorporate a pH-responsive linker between the polysaccharide backbone and the QSI, and to encapsulate CIP via charge-charge interactions of the positively-charged drug with the carboxyl residues of the alginate matrix. In this way, a dual-action release of antibiotic and QSI was designed for the low-pH regions of a biofilm, involving cleavage of the QSI-linker to the alginate matrix and reduced charge-charge interactions between CIP and the polysaccharide as the alginate carboxyl side-chains protonated. When tested in a biofilm model the concomitant release of CIP+QSI from the pH-responsive nanoparticles significantly reduced the viability of the biofilm compared with CIP treatment alone. In addition, the alginate NPs were shown to penetrate deeply into P. aeruginosa biofilms, which we attribute in part to the charges of the NPs and the release of the QSI agent. Finally, we tested the formulation in both a 2D keratinocyte and a 3D ex-vivo skin infection model. The dual-action bio-responsive QSI and CIP release nanoparticles effectively cleared the infection in the latter, suggesting considerable promise for combination therapeutics which prevent biofilm formation as well as effectively killing mature P. aeruginosa biofilms

Passerini chemistries for synthesis of polymer pro-drug and polymersome drug delivery nanoparticles

New materials chemistries are urgently needed to overcome the limitations of existing biomedical materials in terms of preparation, functionality and versatility, and also in regards to their compatibility with biological environments. Here, we show that Passerini reactions are especially suited for the preparation of drug delivery materials, as with relatively few steps, polymers can be synthesized with functionality installed enabling drug conjugation and encapsulation, self-assembly into micellar or vesicular architectures, and with facile attachment triggerable chemistries. The polymers can be made with a variety of building blocks and assemble into nanoparticles, which are rapidly internalized in triple negative breast cancer (TNBC) cells. In addition, the polymers transport drug molecules efficiently through 3D cell cultures, and when designed with chemistries allowing pH-mediated release, exhibit greater efficacy against TNBC cells compared to the parent drug

Biocompatible unimolecular micelles obtained via the Passerini reaction as versatile nanocarriers for potential medical applications

A Passerini three-component polymerization was performed for the synthesis of amphiphilic star-shaped block copolymers with hydrophobic cores and hydrophilic coronae. The degree of polymerization of the hydrophobic core was varied from 5 to 10 repeating units, and the side chain ends were conjugated by performing a Passerini-3CR with PEG-isocyanide and PEG-aldehyde (950 g/mol). The resulting amphiphilic star-shaped block copolymers contained thioether groups, which could be oxidized to sulfones in order to further tune the polarity of the polymer chains. The ability of the amphiphilic copolymers to act as unimolecular micellar encapsulants was tested with the water-insoluble dye Orange II, the water-soluble dye Para Red and the macrolide antibiotic azithromycin. The results showed that the new copolymers were able to retain drug cargo at pH levels corresponding to circulating blood and selectively release therapeutically effective doses of antibiotic as measured by bacterial cell kill. The polymers were also well-tolerated by differentiated THP-1 macrophages in the absence of encapsulated drugs

Synthesis of Passerini-3CR Polymers and Assembly into Cytocompatible Polymersomes

© 2020 The Authors. Published by Wiley-VCH GmbH The versatility of the Passerini three component reaction (Passerini-3CR) is herein exploited for the synthesis of an amphiphilic diblock copolymer, which self-assembles into polymersomes. Carboxy-functionalized poly(ethylene glycol) methyl ether is reacted with AB-type bifunctional monomers and tert-butyl isocyanide in a single process via Passerini-3CR. The resultant diblock copolymer (P1) is obtained in good yield and molar mass dispersity and is well tolerated in model cell lines. The Passerini-3CR versatility and reproducibility are shown by the synthesis of P2, P3, and P4 copolymers. The ability of the Passerini P1 polymersomes to incorporate hydrophilic molecules is verified by loading doxorubicin hydrochloride in P1DOX polymersomes. The flexibility of the synthesis is further demonstrated by simple post-functionalization with a dye, Cyanine-5 (Cy5). The obtained P1-Cy5 polymersomes rapidly internalize in 2D cell monolayers and penetrate deep into 3D spheroids of MDA-MB-231 triple-negative breast cancer cells. P1-Cy5 polymersomes injected systemically in healthy mice are well tolerated and no visible adverse effects are seen under the conditions tested. These data demonstrate that new, biodegradable, biocompatible polymersomes having properties suitable for future use in drug delivery can be easily synthesized by the Passerini-3CR

Passerini chemistries for synthesis of polymer pro-drug and polymersome drug delivery nanoparticles

New materials chemistries are urgently needed to overcome the limitations of existing biomedical materials in terms of preparation, functionality and versatility, and also in regards to their compatibility with biological environments. Here, we show that Passerini reactions are especially suited for the preparation of drug delivery materials, as with relatively few steps, polymers can be synthesized with functionality installed enabling drug conjugation and encapsulation, self-assembly into micellar or vesicular architectures, and with facile attachment triggerable chemistries. The polymers can be made with a variety of building blocks and assemble into nanoparticles, which are rapidly internalized in triple negative breast cancer (TNBC) cells. In addition, the polymers transport drug molecules efficiently through 3D cell cultures, and when designed with chemistries allowing pH-mediated release, exhibit greater efficacy against TNBC cells compared to the parent drug

Passerini multicomponent reaction: a versatile synthesis of nanomedicines for triple negative breast cancer treatment

The Passerini three component reaction (Passerini-3CR) is a multicomponent isocyanide-based reaction, which combines in one-pot and in a straightforward way an aldehyde, an isocyanide and a carboxylic acid into an ester-amide product. The flexibility of the reaction combined with the accessible functionality and the inherent biodegradability of the polymer product therefore offers much potential for new materials applicable in drug delivery and medicinal chemistry. In particular, the high functional group content in the Passerini polymer backbone could be of value in cancer drug delivery systems, as many materials designed for oncology applications lack sufficient drug payload. In this thesis, the focus is on materials for Triple Negative Breast Cancer (TNBC) therapies, as TNBC is an aggressive subtype of breast cancer that has poor patient survival and lack of targeted therapies. Patients are currently treated with taxane or anthracycline-based chemotherapy regiments, which have positive clinical outcomes only in the 30-40% of patients in early stage and poor survival in metastatic patients. The work in this study aims to develop functional biodegradable and biocompatible drug delivery systems for the chemotherapy treatment of TNBC, by exploiting the versatility of the Passerini-3CR. In Chapter 2, amphiphilic diblock copolymers with mPEG 2 and 5 kDa were synthesized via Passerini-3CR and formulated into polymersomes. The vesicles had a colloidally-stabilising and anti-biofouling PEG shell, were found to be stable in blood-mimicking pH conditions and to have low critical aggregation concentrations. The cytocompatibility of Passerini copolymers was verified in TNBC cells and non-cancerous human mammary epithelial cells. The internalization of fluorescently labelled Passerini polymersomes was investigated in 2D and 3D in TNBC cells and was found to be concentration dependent and endocytosis mediated. Doxorubicin was loaded in the polymersomes via the remote transmembrane pH gradient method. The efficacy of the doxorubicin-loaded polymersomes was found to be comparable with the free drug, thus indicating efficient release of doxorubicin from the delivery system. Furthermore, the drug was found to be selectively released under endolysosomal mimicking pH acidic conditions and retained under blood mimicking pH conditions. In Chapter 3, amphiphilic triblock copolymers were synthesized via Passerini-3CR and decorated on the surface with the cMET binding peptide as a targeting agent. The cMET receptor has been reported to be overexpressed in TNBC, therefore the polymersomes decoration with the cMET binding peptide was aimed to improve the actively targeted polymersomes internalization in TNBC cells over non-cancerous cells. Uptake studies were performed in TNBC cells in 2D and 3D culture, and the actively targeted polymersomes were found to be internalized to a higher extent than the untargeted analogues. Finally, in Chapter 4, the versatility of the Passerini-3CR was exploited for the synthesis of diblock amphiphilic copolymers with a high density of alkene pendant groups. These groups were oxidised by ozonolysis into aldehydes, which were quantitatively conjugated to the amino group of doxorubicin via the pH-responsive amine linker. The obtained doxorubicin-polymer conjugates were assembled into solid nanoparticles and tested in TNBC cells. The prodrug nanoparticle was found to be more cytotoxic than the free drug, suggesting the potential of this formulation of improving the doxorubicin activity, by performing a constant and continuous drug release in the endolysosomal cellular compartment. Moreover, the drug was found to be selectively released under endolysosomal mimicking pH conditions and to be retained under blood pH mimicking conditions. Overall, these results demonstrate that the Passerini-3CR is a promising synthetic route to generate polymeric nanomedicines that might in future improve the chemotherapy treatment of TNBC

Passerini multicomponent reaction: a versatile synthesis of nanomedicines for triple negative breast cancer treatment

The Passerini three component reaction (Passerini-3CR) is a multicomponent isocyanide-based reaction, which combines in one-pot and in a straightforward way an aldehyde, an isocyanide and a carboxylic acid into an ester-amide product. The flexibility of the reaction combined with the accessible functionality and the inherent biodegradability of the polymer product therefore offers much potential for new materials applicable in drug delivery and medicinal chemistry. In particular, the high functional group content in the Passerini polymer backbone could be of value in cancer drug delivery systems, as many materials designed for oncology applications lack sufficient drug payload. In this thesis, the focus is on materials for Triple Negative Breast Cancer (TNBC) therapies, as TNBC is an aggressive subtype of breast cancer that has poor patient survival and lack of targeted therapies. Patients are currently treated with taxane or anthracycline-based chemotherapy regiments, which have positive clinical outcomes only in the 30-40% of patients in early stage and poor survival in metastatic patients. The work in this study aims to develop functional biodegradable and biocompatible drug delivery systems for the chemotherapy treatment of TNBC, by exploiting the versatility of the Passerini-3CR. In Chapter 2, amphiphilic diblock copolymers with mPEG 2 and 5 kDa were synthesized via Passerini-3CR and formulated into polymersomes. The vesicles had a colloidally-stabilising and anti-biofouling PEG shell, were found to be stable in blood-mimicking pH conditions and to have low critical aggregation concentrations. The cytocompatibility of Passerini copolymers was verified in TNBC cells and non-cancerous human mammary epithelial cells. The internalization of fluorescently labelled Passerini polymersomes was investigated in 2D and 3D in TNBC cells and was found to be concentration dependent and endocytosis mediated. Doxorubicin was loaded in the polymersomes via the remote transmembrane pH gradient method. The efficacy of the doxorubicin-loaded polymersomes was found to be comparable with the free drug, thus indicating efficient release of doxorubicin from the delivery system. Furthermore, the drug was found to be selectively released under endolysosomal mimicking pH acidic conditions and retained under blood mimicking pH conditions. In Chapter 3, amphiphilic triblock copolymers were synthesized via Passerini-3CR and decorated on the surface with the cMET binding peptide as a targeting agent. The cMET receptor has been reported to be overexpressed in TNBC, therefore the polymersomes decoration with the cMET binding peptide was aimed to improve the actively targeted polymersomes internalization in TNBC cells over non-cancerous cells. Uptake studies were performed in TNBC cells in 2D and 3D culture, and the actively targeted polymersomes were found to be internalized to a higher extent than the untargeted analogues. Finally, in Chapter 4, the versatility of the Passerini-3CR was exploited for the synthesis of diblock amphiphilic copolymers with a high density of alkene pendant groups. These groups were oxidised by ozonolysis into aldehydes, which were quantitatively conjugated to the amino group of doxorubicin via the pH-responsive amine linker. The obtained doxorubicin-polymer conjugates were assembled into solid nanoparticles and tested in TNBC cells. The prodrug nanoparticle was found to be more cytotoxic than the free drug, suggesting the potential of this formulation of improving the doxorubicin activity, by performing a constant and continuous drug release in the endolysosomal cellular compartment. Moreover, the drug was found to be selectively released under endolysosomal mimicking pH conditions and to be retained under blood pH mimicking conditions. Overall, these results demonstrate that the Passerini-3CR is a promising synthetic route to generate polymeric nanomedicines that might in future improve the chemotherapy treatment of TNBC