Synthesis of zwitterionic-functionalized conjugated nanoparticles for targeted drug delivery applications

Polymeric Nanoparticles (NPs) represent a promising pharmacological tool, since their structure can be modified to obtain: i) encapsulation and controlled release of a wide range of active compounds, ranging from small molecules to siRNA or oligonucleotides; ii) selective cell targeting, thus allowing precise drug delivery to the desired site of action. A powerful strategy to achieve selectivity of uptake in specific cell types is to conjugate the nanoparticles to a ligand specific for receptors expressed by the target cell type. This offers the advantage of a potentially improved drug efficacy with limited side effects and toxicity. Polymeric nanoparticles in a range of 20–100 nm have a high potential for in vivo applications, due to their ability to circulate in the blood for a long period of time. In fact, this size range allows to avoid renal and lymphatic clearance, to prevent opsonization and at the same time improves the internalization by cells. In this work we address the synthesis by reversible addition-fragmentation chain transfer (RAFT) of biodegradable, zwitterionic-based nanoparticles. This Zwitterionic nanoparticles act as super non-fouling surfaces that prevent protein adsorption from complex biological media. The nanoparticles were functionalized with different numbers of selective ligands through click chemistry; different dimensions were synthetized changing the length of the hydrophobic part. In vitro studies were performed to evaluate the uptake of functionalized nanoparticles

The Combination Of Rop And Raft Polymerization For The Synthesis Of Polymeric Nanoparticles

Polymeric nanoparticles (NPs) are colloids in the nanometric size that find application in several field, such as optics, coating and medicine. In this latter case, they are used as drug delivery systems for different therapeutics ranging from lipophilic drugs to oligonucleotides. These nano-colloids are generally made up of polyesters as long as they are able to degrade into safe and easy removable compounds, such as lactic acid and hydroxycaproic acid. In this work, starting from a geometrical model developed for the synthesis of NPs with the same NP size and different molecular weight (MW) block copolymers1, a method to independently control the main characteristics of biodegradable NPs stabilized by highly hydrophilic polymers has been developed and here presented. The method consists in the synthesis of block-copolymers with a brush-like structure via the combination of two living polymerizations: the ring opening polymerization (ROP) and the reversible addition-fragmentation chain transfer (RAFT) polymerization. A library of block copolymers has been produced and self-assembled into water to generate NPs with different size and block-copolymer MWs. As long as these NPs are intended for biomedical applications, the degradation behavior of these colloids has been studied and correlated with the structure of the lipophilic part of the block copolymer. It has been found that the number of the lactone units and their geometrical disposition in the block copolymers impact the degradation behavior of the NPs they are composed of. Thanks to this novel method, it is possible to synthesize NPs with the same size, but with different degradation time. References 1. Palmiero, U. C.; Agostini, A.; Gatti, S.; Sponchioni, M.; Valenti, V.; Brunel, L.; Moscatelli, D., Raft macro-surfmers and their use in the ab initio raft emulsion polymerization to decouple nanoparticle size and polymer molecular weight. Macromolecules 2016, 49 (22), 8387-8396

Polyester-based excipients to formulate lipophilic drugs into nanoparticles directly at the bed of the patient

In recent decades there has been an increased interest in polymeric nanoparticles as drug delivery systems thanks to their several advantages, such as continuous maintenance of drug levels in a therapeutically desirable range, and reduction of harmful side effects. These nano-colloids are generally made up of polyesters as long as they are able to easily degrade into the body. However, NP production is often a process that requires complex microfluidic devices. In addition, expensive purification steps are necessary to eliminate the unloaded drug and the high amount of organic solvent used in the NP production step. In the end, a lyophilization step is general adopted to assure a good shelf-life of the final product. All the above-mentioned steps hamper the cost-effective use of a re-formulation of the same therapeutic agent and, in turn, reduce the availability of these treatments among the patient population. For this reason, in this work, a novel NP production protocol that consists only in the use of a syringe and a needle without the need of subsequent purification and freeze-drying steps has been developed. This has been possible by the optimization of the hydrophilic/lipophilic balance of block-copolymers that are able to directly self-assemble in water. The additional degree of freedom necessary for this optimization was introduced via the synthesis of these materials thorough the combination of the reversible addition-fragmentation chain transfer (RAFT) polymerization and the ring opening polymerization (ROP). The NPs has been used to formulate Trabectedin (ET-743), a widely adopted anticancer therapeutic known for its local adverse effect. The pharmacokinetic behavior, antitumor activity and toxicity of this novel NP-based formulation has been compared to the commercially available formulation Yondelis®. NPs have shown the ability to retain the drug into circulation for a longer time in the blood stream compared to the free ET-743 allowing to considerably reduce the local toxic effects. In addition, the shift of the NP preparation step from a specialist to the final user allows to avoid all the purifications and post-processing steps necessary to assure a good shelf-life of the product. In this way, a ET-743 formulation less toxic than the commercially available Yondelis® can be produced at a competitive price taking also into account that this expensive drug is not lost in any of the NP production steps here adopted. In order to prove the versatility of this novel technology, Paclitaxel (PTX), an anticancer therapeutic that it usually formulated with a toxic surfactant (Chremophor EL), have been also formulated into this NPs. In this way, a novel PTX formulation can be produced at a lower cost compared to the ones already approved and present into the market. In particular, it has shown the same advantage in reduction of the toxicity given by the elimination of the Chremophor EL (e.g in Abraxane® and Genexol®)

Poly(HPMA)-based copolymers with biodegradable side chains able to self-assemble into nanoparticles

Poly(N-(2-Hydroxypropyl) methacrylamide) (poly(HPMA)) is gaining pharmaceutical attention in replacement to PEG as a hydrophilic stabilizer for polymer nanoparticles (NPs) devoted to systemic administration.[1] This is due to its biocompatibility, prolonged circulation time and, compared to PEG, to the avoidance of allergic reactions and of the accelerated blood clearance effect.[2, 3] In this work, a lipophilic HPMA-based macromonomer with a predetermined and controllable structure is synthesized for the first time attaching a short oligo(caprolactone) chain obtained via Ring Opening Polymerization (ROP) to the HPMA using a succinic acid unit as a spacer. This biodegradable monomer (hereinafter HPMA-CL) was then used to synthesize well-defined amphiphilic block copolymers comprising a hydrophilic poly(HPMA) block and a hydrophobic poly(HPMA-CL) segment via Reversible Addition-Fragmentation Transfer (RAFT) polymerization. The combination of ROP and RAFT allows the production of a library of polymers with a predetermined and controlled structure that are able to self-assemble in water into biodegradable NPs with different size. In particular, such NPs are designed to degrade in aqueous environment into completely water soluble poly(HPMA), with a molecular weight that is below the critical threshold for the renal excretion. This is a very important feature since it allows to avoid polymer accumulation into the body once the NPs are injected.[4] The degradation time is a function of the number of caprolactone units in the HPMA-CL macromonomer and of its degree of polymerization in the NP forming copolymer. Then, the polymer structure can be adjusted to obtain the desired degradation time. Finally, the possibility for such nanoparticles to physically incorporate and mediate the release of a lipophilic antineoplastic drug was evaluated in the case of Trabectedin. The formulation proved to be biocompatible and to sustainedly release the drug for up to 24 hours. Please click Additional Files below to see the full abstract

Microfluidic and nanotechnology based assays for the development of safe biopharmaceuticals

Protein stability towards aggregation represents a potential challenge for the production and administration of pharmaceuticals. In particular, aggregation can compromise the developability and shelf-life of the products, with consequences for yield and safety, respectively. In this work, we discuss two novel approaches for the analysis of the stability of protein formulations: (1) A microfluidic diffusion-sizing platform to analyze protein sizes and interactions at high protein concentration directly in the solution state with minimal perturbation of the sample. The limited dilution of the sample during the analysis and the possibility to characterize properties directly in the solution state make the technique suitable for the analysis of heterogeneous solutions of proteins under dynamic equilibrium. We show how the platform represents an attractive tool for the analysis of sizes and interactions of proteins in both diluted and high-concentration solutions during development, manufacturing, and formulation. (2) A highly controlled assay of surface-induced protein aggregation based on nanoparticles. Protein aggregation is often due to heterogeneous nucleation events occurring at interfaces, including air/water interface, impurities and leachable particles. However, the development of screening tools against surface aggregation has been hindered by the difficulty in generating a controlled amount of surface stress in the formulation as well as in decoupling the surface effect from the contribution of hydrodynamic flows. In our assay, we leverage the flexibility of polymer chemistry to finely tune the properties and amount of surfaces provided by the nanoparticles, inducing aggregation of soluble peptides and proteins, including antibodies, in a time scale of a few hours. This platform represents i) an attractive tool for fundamental studies of heterogeneous nucleation events under stagnant and flow conditions, and ii) a high-throughput screening assay of the effect of intrinsic and extrinsic variables on protein stability towards interface-induced aggregation. Please click Additional Files below to see the full abstract

The RAFT polymerization and its application to aqueous dispersed systems

The reversible addition-fragmentation chain transfer polymerization is one of the most important techniques to control the molecular weight and the chain distribution of the polymers made via radical chemistry. Its application to the aqueous dispersed systems is an important step to improve the control of relevant polymers due to the advantages in the control of the reaction and to avoid the environmental issues related to the use of organic solvents. In this review, a brief overview on different techniques used to perform a well-controlled RAFT polymerization in aqueous dispersed systems is critically revised

HPMA-PEG Surfmers and Their Use in Stabilizing Fully Biodegradable Polymer Nanoparticles

The most adopted methods to produce polymer nanoparticles (NPs) for medical and pharmaceutical applications use surfactants that are toxic and physically adsorbed to the NPs, with the risk of desorption and insurgence of side effects. A valid alternative is represented by the use of surfmers, reactive surfactants that are chemically linked to the polymer chains, thus avoiding the release of toxic species. In this case, the lack of biodegradable surfmers introduces the concern of polymer accumulation into the body. In this work, biodegradable, poly(ethylene glycol) (PEG)ylated N-(2-hydroxypropyl) methacrylamide (HPMA) based surfmers are synthesized and used to stabilize lipophilic NPs. In particular, the NP core is made from a macromonomer comprising a poly(lactic acid) (PLA) chain functionalized with HPMA double bond. The NP forming polymer chains are then constituted by a uniform poly(HPMA) backbone that is biocompatible and water soluble and hydrolysable PEG and PLA pendants assuring the complete degradability of the polymer. The stability provided to these NPs by the synthesized surfmers is studied in the cases of both emulsion free radical polymerization and solution free radical polymerization followed by the flash nanoprecipitation of the obtained amphiphilic copolymers

Zwitterionic Polyester-Based Nanoparticles with Tunable Size, Polymer Molecular Weight, and Degradation Time

Biodegradable polymer nanoparticles are an important class of materials used in several applications for their unique characteristics. In particular, the ones stabilized by zwitterionic materials are gaining increased interest in medicine as alternative to the more common ones based on poly(ethylene glycol) thanks to their superior stability and ability to avoid both the accelerated blood clearance and allergic reactions. In this work, a novel class of zwitterionic based NPs has been produced, and a method to independently control the nanoparticle size, degradation time, and polymer molecular weight has been developed and demonstrated. This has been possible by the synthesis and the fine-tuning of zwitterionic amphiphilic block copolymers obtained via the combination of ring-opening polymerization and reversible addition-fragmentation chain transfer polymerization. The final results showed that when two block copolymers contain the same number of caprolactone units, the one with longer oligoester lateral chains degrades faster. This phenomenon is in sharp contrast with the one seen so far for the common linear polyester systems where longer chains result in longer degradation times, and it can be used to better tailor the degradation behavior of the nanoparticles

RAFT copolymerization of oppositely charged monomers and its use to tailor the composition of nonfouling polyampholytes with an UCST behaviour

Synthetic polyampholytes are attracting significant interest due to the possibility that they offer of combining oppositely charged repeating units in the same copolymer chain. This peculiar structure makes them appealing for the understanding of biological processes such as protein folding and DNA condensation as well as for application as pH- and salt-responsive gels. In addition, the alternation at the molecular level of charges with opposite signs holds promise in avoiding the fouling of proteins, bacteria, and marine organisms. Indeed, polymer reaction engineering assumes an important role in ensuring such charge alternation and in turn the efficacy of the polyampholyte coating. In this work, the reversible addition-fragmentation chain transfer (RAFT) copolymerization of the electrolyte monomers 3-sulfopropyl methacrylate potassium salt (anionic, SPMAK) and [2-(methacryloyloxy)ethyl]trimethylammonium chloride (cationic, MADQUAT) was studied in detail to highlight the influence of both the monomer and chain transfer agent (CTA) concentrations on the polymerization rate and copolymer composition. By analysing the residual monomer mixture composition via in situ nuclear magnetic resonance (1H NMR), the reactivity ratios for the two monomers were calculated. Interestingly, the values obtained in the case of RAFT copolymerization (i.e. rSPMAK = 0.51 ± 0.03 and rMADQUAT = 0.31 ± 0.03) are valid in the case of a conventional free-radical copolymerization under similar conditions. The optimized polyampholytes showed interesting aqueous properties, including an upper critical solution temperature (UCST), which was studied as a function of the salt concentration and polymer molecular weight. Finally, as a proof of concept, the efficacy of the synthesized polyampholytes as nonfouling coatings was assessed in the case of A375-P cells

RAFT macro-surfmers and their use in the ab initio RAFT emulsion polymerization to decouple nanoparticle size and polymer molecular weight

Polymeric nanoparticles (NPs) are highly engineered nanoemulsions with applications in several fields. The control over NP surface chemistry, size (Dn), and molecular weight (MW) of the polymer they are made up of plays a paramount role in the optimization of their end-use performance. In this work, the theoretical basis to decouple the dependence between the NP Dn and MW has been presented, and an operative way has been demonstrated via ab initio reversible addition-fragmentation chain transfer (RAFT) emulsion polymerization (AIREP), a "living" heterogeneous process that adopts RAFT macro-surfmers: macromolecular chain transfer agents (CTA) produced via RAFT polymerization of amphiliphic monomers, such as surfmers. The possibility of obtaining the desired length of the lipophilic block or the length of the whole block copolymer and the NP Dn by choosing the correct length of the RAFT macro-surfmer has been assessed. It has been discovered that a wide range of Dn and MW can be achieved, but not very big NPs with very low copolymer MW; this limit is consistent with the physical and geometric constraints of the system