Adrian Sulistio

Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers

With the recent emergence of reports on resistant Gram-negative ‘superbugs’, infections caused by multidrug-resistant (MDR) Gram-negative bacteria have been named as one of the most urgent global health threats due to the lack of effective and biocompatible drugs. Here, we show that a class of antimicrobial agents, termed ‘structurally nanoengineered antimicrobial peptide polymers’ (SNAPPs) exhibit sub-μM activity against all Gram-negative bacteria tested, including ESKAPE and colistin-resistant and MDR (CMDR) pathogens, while demonstrating low toxicity. SNAPPs are highly effective in combating CMDR Acinetobacter baumannii infections in vivo, the first example of a synthetic antimicrobial polymer with CMDR Gram-negative pathogen efficacy. Furthermore, we did not observe any resistance acquisition by A. baumannii (including the CMDR strain) to SNAPPs. Comprehensive analyses using a range of microscopy and (bio)assay techniques revealed that the antimicrobial activity of SNAPPs proceeds via a multimodal mechanism of bacterial cell death by outer membrane destabilization, unregulated ion movement across the cytoplasmic membrane and induction of the apoptotic-like death pathway, possibly accounting for why we did not observe resistance to SNAPPs in CMDR bacteria. Overall, SNAPPs show great promise as low-cost and effective antimicrobial agents and may represent a weapon in combating the growing threat of MDR Gram-negative bacteria.

Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells

Amino acid-based core cross-linked star (CCS) polymers (poly(L-lysine)(arm)poly(L-cystine)(core)) with peripheral allyl functionalities were synthesized by sequential ring-opening polymerization (ROP) of amino acid N-carboxyanhydrides (NCAs) via the arm-first approach, using N-(trimethylsilyl)allylamine as the initiator. Subsequent functionalization with a poly(ethylene glycol) (PEG)-folic acid conjugate via thiol-ene click chemistry afforded poly(PEG-b-L-lysine)(arm)poly(L-cystine)(core) stars with outer PEG coronas decorated with folic acid targeting moieties. Similarly, a control was prepared without folic acid, using just PEG. A fluorophore was used to track both star polymers incubated with breast cancer cells (MDA-MB-231) in vitro. Confocal microscopy and flow cytometry revealed that the stars could be internalized into the cells, and higher cell internalization was observed when folic acid moieties were present. Cytotoxicity studies indicate that both stars are nontoxic to MDA-MB-231 cells at concentrations of up to 50 μg/mL. These results make this amino acid-based star polymer an attractive candidate in targeted drug delivery applications including chemotherapy.

Chemical Cross-Linking Gelatin with Natural Phenolic Compounds as Studied by High-Resolution NMR Spectroscopy

Cross-linking gelatin with natural phenolic compound caffeic acid (CA) or tannic acid (TA) above pH 9 resulted in formation of insoluble hydrogels. The cross-linking reactivity was controlled by variation of pH, the concentration of the gelatin solution, or the amount of CA or TA used in the reaction. The cross-linking chemistry was studied by high-resolution NMR technique in both solution and solid state via investigation on small molecular model systems or using (13)C enriched caffeic acid (LCA) in the reaction with gelatin. Direct evidence was obtained to confirm the chemical reactions occurring between the phenolic reactive sites of the phenolic compounds and the amino groups in gelatin to form C-N covalent bonds as cross-linking linkages in gelatin matrix. The cross-linked network was homogeneous on a scale of 2-3 nm. The cross-linking resulted in a significant decrease in the molecular mobility of the hydrogels, while the modulus of the films remained at high values at high temperatures.

Chemical modification of gelatin by a natural phenolic cross-linker, tannic acid.

Chemical modification of gelatin by a natural phenolic compound tannic acid (TA) at pH 8 was studied, and the properties of the modified gelatin materials were examined. The cross-linking effect was predominant when the TA content was lower, resulting in the formation of a partially insoluble cross-link network. The cross-linking structure was stable even under boiling, and the protein matrix became rigid, whereas the mechanical properties were enhanced. An effective cross-linking effect on gelatin matrix was achieved when the amount of TA was around 3 wt %. Further increase in the TA content enhanced the grafting and branching reactions between gelatin and TA in conjunction with the hydrogen bonding between gelatin and TA molecules. These effects produced an increase in molecular mobility of gelatin matrix, and the materials displayed a behavior similar to that of plasticized protein materials.

Development of functional amino acid-based star polymers

Highly functionalized core cross-linked star (CCS) polymers composed entirely of naturally-occurring amino acids were prepared via the sequential ring-opening polymerisation (ROP) of amino acidN-carboxyanhydride (NCA) derivatives in a facile one-pot, arm-first strategy. The formation of the star polymers was investigated using side-chain protected poly(e-Z-L-lysine) (PZLL) and poly(γ-benzyl-L-glutamic acid) (PBGA) macroinitiators with various molecular weights in combination with a cystine NCA cross-linker to afford poly(e-Z-L-lysine)armspoly(L-cystine)core (PZLLarmsPLCcore) and poly(γ-benzyl-L-glutamic acid)armspoly(L-cystine)core (PBLGarmsPLCcore) stars, respectively. As the cross-linker to macroinitiator ratio or macroinitiator molecular weights were increased the molecular weights, average number of arms and core size of the resulting stars also increased. Core-isolated NCA moieties remaining after star formation provided a facile approach to core-functionalization with primary amines bearing different functionalities, including aminomethyl pyrene, propargylamine and hexylamine. UV-vis spectroscopic analysis of PZLLarmsPLCcore and PBLGarmsPLCcore stars core-functionalised with aminomethyl pyrene provided high loadings of 240 and 128 mol/mol stars, respectively. Furthermore, stars with alkyne functionalized cores were capable of undergoing further click reactions with azido derivatives, demonstrating the accessibility of the core-isolated moieties. Deprotection of the PZLLarmsPLCcore and PBLGarmsPLCcore stars yielded water soluble CCS polymers with poly(L-lysine) and poly(L-glutamic acid) arms, respectively, and functionalised cores. In addition, direct hydrazinolysis of the PBLGarmsPLCcore star provided hydrazide functionalities along the arms, which allow for conjugation of drug molecules via pH sensitive hydrazone linkers. These results open up exciting opportunities for the development of star polymer drug delivery systems, whereby a lack of functionality, biocompatibility and biodegradability are often limiting factors.

Peptide-Based Star Polymers: The Rising Star in Functional Polymers

Peptide-based star polymers show great potential as the next-generation of functional polymers due to their structure-related properties. The peptide component augments the polymer’s properties by introducing biocompatible and biodegradable segments, and enhancing their functionalities and structural ordering, which make peptide-based star polymers an attractive candidate in the field of nanomedicine. This article provides a brief summary of the recent developments of peptide-based star polymers synthesised from 2009 onwards. It is evident that the studies conducted so far have only started to uncover the true potential of what these polymers can achieve, and with continued research it is anticipated that peptide-based star polymers will be realised as versatile platforms applicable to broader fields of study, including drug delivery, tissue engineering, biocoatings, bioimaging, and self-directing templating agents.

Stabilization of peptide-based vesicles via in situ oxygen-mediated cross-linking

Reversible vesicles from poly(L-glutamic acid)(65) -block-poly[(L-lysine)-ran-(L-3,4-dihydroxyphenylalanine)](75) [PLGA(65)-b-P(LL-r-DOPA)(75)] block copolypeptide adopt different configurations depending on the surrounding pH. At pH = 3, AFM and TEM images show ellipsoidal morphologies, whereas at pH = 12 both TEM and AFM reveal the formation of hollow vesicles. At pH = 12, the P(LL-r-DOPA) block forms the internal layer of the vesicle shell and the subsequent oxygen-mediated oxidation of the phenolic groups of the DOPA lead to the formation of quinonic intermediates, which undergo intermolecular dimerization to stabilize the vesicles via in situ cross-linking. Consequently, the vesicles maintain their shape even when the pH is reversed back to 3, as confirmed by AFM and TEM.

Peptide-Based Star Polymers as Potential siRNA Carriers

16- and 32-arm star polymers were synthesised using poly(amido amine) (PAMAM) dendrimers as multifunctional initiators for the ring-opening polymerisation (ROP) of ϵ-Z-l-lysine N-carboxyanhydride (Lys NCA) via the core-first approach. The resulting star polymers were subsequently post-functionalised with poly(ethylene glycol) (PEG) via carbodiimide coupling, potentially improving the biodistribution of the stars in vivo. De-protection of the carboxybenzyl (Cbz)-protected star arms yielded water-soluble cationic poly(l-lysine) (PLL) star polymers with hydrodynamic radii ranging from 2.0 to 3.3 nm. Successful complexation of the PLL star polymers with double-stranded oligodeoxynucleotides (ODNs)—a mimic for small interfering RNA (siRNA)—was achieved at a nitrogen-to-phosphate (N/P) ratio of 5. Cell viability studies using HEK293T cells indicated the ‘safe’ concentration for these polymers is within a suitable window for the delivery of siRNA therapeutics.

Assembly of Free-Standing Polypeptide Films via the Synergistic Combination of Hyperbranched Macroinitiators, the Grafting-From Approach, and Cross-Chain Termination

Cross-linked polypeptide-based films are fabricated via a novel and robust method employing surface-initiated ring opening polymerization of α-amino acid N-carboxyanhydrides (NCA-ROP). The judicious combination of amine-based hyperbranched macroinitiators and benzyl ester-protected NCA derivatives promotes network formation by cross-chain terminations, which allows the formation of stable cross-linked peptide-based capsules in a one-pot system.

Azobenzene-Functionalised Core Cross-Linked Star Polymers and their Host–Guest Interactions

Water-soluble poly(2-hydroxyethyl acrylate) (PHEA)-based core cross-linked star polymers were efficiently synthesised with high macroinitiator-to-star-conversion (>95 %) in a one-pot system via single electron transfer-living radical polymerisation. The star polymers display excellent water solubility and the pendant hydroxyl groups provide a platform for facile post-functionalisation with various molecules. In demonstrating this, a photo-isomerisable molecule, 4-(phenylazo)benzoic acid was conjugated onto the preformed stars through partial esterification of the available hydroxyl groups (5–20 %). The azobenzene functionalised stars were subsequently employed to form reversible inclusion complexes with α-cyclodextrin.