L-DOPA functionalized, multi-branched gold nanoparticles as brain-targeted nano-vehicles.

The blood-brain barrier (BBB) is a protective endothelial barrier lining the brain microvasculature which prevents brain delivery of therapies against brain diseases. Hence, there is an urgent need to develop vehicles which efficiently penetrate the BBB to deliver therapies into the brain. The drug L-DOPA efficiently and specifically crosses the BBB via the large neutral amino acid transporter (LAT)-1 protein to enter the brain. Thus, we synthesized L-DOPA-functionalized multi-branched nanoflower-like gold nanoparticles (L-DOPA-AuNFs) using a seed-mediated method involving catechols as a direct reducing-cum-capping agent, and examined their ability to cross the BBB to act as brain-penetrating nanovehicles. We show that L-DOPA-AuNFs efficiently penetrate the BBB compared to similarly sized and shaped AuNFs functionalized with a non-targeting ligand. Furthermore, we show that L-DOPA-AuNFs are efficiently internalized by brain macrophages without inducing inflammation. These results demonstrate the application of L-DOPA-AuNFs as a non-inflammatory BBB-penetrating nanovehicle to efficiently deliver therapies into the brain.

Non-Isocyanate Polyurethane Soft Nanoparticles Obtained by Surfactant-Assisted Interfacial Polymerization.

Polyurethanes (PUs) are considered ideal candidates for drug delivery applications due to their easy synthesis, excellent mechanical properties, and biodegradability. Unfortunately, methods for preparing well-defined PU nanoparticles required miniemulsion polymerization techniques with a nontrivial control of the polymerization conditions due to the inherent incompatibility of isocyanate-containing monomers and water. In this work, we report the preparation of soft PU nanoparticles in a one-pot process using interfacial polymerization that employs a non-isocyanate polymerization route that minimizes side reactions with water. Activated pentafluorophenyl dicarbonates were polymerized with diamines and/or triamines by interfacial polymerization in the presence of an anionic emulsifier, which afforded non-isocyanate polyurethane (NIPU) nanoparticles with sizes in the range of 200-300 nm. Notably, 5 wt % of emulsifier was required in combination with a trifunctional amine to achieve stable PU dispersions and avoid particle aggregation. The versatility of this polymerization process allows for incorporation of functional groups into the PU nanoparticles, such as carboxylic acids, which can encapsulate the chemotherapeutic doxorubicin through ionic interactions. Altogether, this waterborne synthetic method for functionalized NIPU soft nanoparticles holds great promise for the preparation of drug delivery nanocarriers.

Overcoming multidrug resistance in microbials using nanostructures self-assembled from cationic bent-core oligomers.

Novel cationic molecules based on rigid terephthalamide-bisurea cores flanked by imidazolium moieties are described. In aqueous media, these compounds self-assemble into supramolecular nanostructures with distinct morphologies. The compound with optimal hydrophilic/hydrophobic balance displays potent antimicrobial activity and high selectivity towards clinically-isolated MRSA without inducing drug-resistance. These self-assembled cationic antimicrobial nanostructures show promise for the prevention and treatment of multidrug-resistant infections.

Free-Standing Hierarchically Porous Silica Nanoparticle Superstructures: Bridging the Nano- to Microscale for Tailorable Delivery of Small and Large Therapeutics.

Nanoscale colloidal self-assembly is an exciting approach to yield superstructures with properties distinct from those of individual nanoparticles. However, the bottom-up self-assembly of 3D nanoparticle superstructures typically requires extensive chemical functionalization, harsh conditions, and a long preparation time, which are undesirable for biomedical applications. Here, we report the directional freezing of porous silica nanoparticles (PSiNPs) as a simple and versatile technique to create anisotropic 3D superstructures with hierarchical porosity afforded by microporous PSiNPs and newly generated meso- and macropores between the PSiNPs. By varying the PSiNP building block size, the interparticle pore sizes can be readily tuned. The newly created hierarchical pores greatly augment the loading of a small molecule-anticancer drug, doxorubicin (Dox), and a large macromolecule, lysozyme (Lyz). Importantly, Dox loading into both the micro- and meso/macropores of the nanoparticle assemblies not only gave a pore size-dependent drug release but also significantly extended the drug release to 25 days compared to a much shorter 7 or 11 day drug release from Dox loaded into either the micro- or meso/macropores only. Moreover, a unique temporal drug release profile, with a higher and faster release of Lyz from the larger interparticle macropores than Dox from the smaller PSiNP micropores, was observed. Finally, the formulation of the Dox-loaded superstructures within a composite hydrogel induces prolonged growth inhibition in a 3D spheroid model of pancreatic ductal adenocarcinoma. This study presents a facile modular approach for the rapid assembly of drug-loaded superstructures in fully aqueous environments and demonstrates their potential as highly tailorable and sustained delivery systems for diverse therapeutics.

Injectable Hydrogels Prepared Using Novel Synthetic Short Peptides with Defined Structure and Gelatin as Scaffolds to Support Cell and Tissue Growth.

Animal-derived basement-membrane matrices such as Geltrex are used to grow cells and tissues. Particularly, these are commonly applied to support tumor growth in animals for cancer research. However, a material derived from an animal source has an undefined composition, and may thus have unavoidable batch-to-batch variation in properties. To overcome these issues, a series of synthetic short peptides to form hydrogels is designed in combination with gelatin to promote cell adhesion and growth. The peptides have sequences of (X1Y1X2Y2)2 , where X1 and X2 are hydrophobic residues, while Y1 and Y2 are hydrophilic residues. The peptides spontaneously fold and self-assemble into a ß-sheet secondary structure upon contact with salts, and then aggregate to form hydrophilic networks of hydrogels. Hybrid hydrogels formed by mixing the peptide IEVEIRVK (IVK8) with gelatin are injectable and enzymatically degradable. The hybrid hydrogels at optimal compositions support SW480 and HepG2 tumor spheroid growth in vitro as effectively as Geltrex. More importantly, the peptide/gelatin hydrogels support tumor growth in a SW480 human colorectal adenocarcinoma xenograft mouse model. Altogether, the results illustrate that the synthetic peptide/gelatin hybrid hydrogel is a promising scaffold that can be used to support cell and tissue growth both in vitro and in vivo.

Antimicrobial polycarbonates: investigating the impact of balancing charge and hydrophobicity using a same-centered polymer approach.

Biodegradable antimicrobial polymers are a promising solution for combating drug resistant microbes. When designing these materials, the balance between charge and hydrophobicity significantly affects the antimicrobial activity and selectivity toward microbes over mammalian cells. Furthermore, where the charge and hydrophobicity is located on the molecules has also proven to be significant. A series of antimicrobial homopolymer polycarbonates were synthesized, where the hydrophobic/hydrophilic balance was controlled by varying the spacer between the charged quaternary ammonium moiety and the polymer backbone (a "same-centered" structure where the hydrophobic moiety is directly attached to the charged moiety). These homopolymers were active against all microbes tested but depending on the spacer length some hemolytic activity was observed. To reduce the polymer hemolytic activity we systematically varied the polymer composition by copolymerizing the different monomers used in the "same center" homopolymers. By maintaining charge on each repeat unit but copolymerizing monomers having varied hydrophobic side chain lengths, polymers with high activity and selectivity were achieved. In addition, these macromolecules act via a membrane disruption mechanism, making them less likely to induce resistance.

Rationally designed α-helical broad-spectrum antimicrobial peptides with idealized facial amphiphilicity.

A series of 12-amino acid peptide analogs is designed using point mutation strategy based on an α-helical peptide template. The first mutation in the series, KL12, has an idealized facial amphiphilicity. Subsequent mutations are performed to increase hydrophobic or cationic contents. Idealized facial amphiphilicity show enhanced antimicrobial activity and selectivity against most of the tested microbes. Increasing hydrophobic contents further enhance antimicrobial potency; however, selectivity of the most hydrophobic analog is impaired due to non-specific interactions with mammalian cell membrane. This study demonstrates that facial amphiphilicity and hydrophobic content are strongly correlated with antimicrobial activity and selectivity of antimicrobial peptides.

Galactose-functionalized cationic polycarbonate diblock copolymer for targeted gene delivery to hepatocytes.

To mediate selective gene delivery to hepatocytes via the asialoglycoprotein receptors (ASGP-Rs), we designed and synthesized well-defined and narrowly dispersed galactose- and glucose-functionalized cationic polycarbonate diblock copolymers (designated as Gal-APC and Glu-APC, respectively) using organocatalytic ring-opening polymerization of functionalized carbonate monomers, with a subsequent quaternization step using bis-tertiary amines to confer quaternary and tertiary amines for DNA binding and endosomal buffering, respectively. The sugar-functionalized diblock copolymers effectively bound and condensed DNA to form positively charged nanoparticles (<100 nm in diameter and ≈30 mV zeta-potential) that were stable under high physiological salt conditions. In comparison to the control Glu-APC/DNA complexes, Gal-APC/DNA complexes mediated significantly higher gene expression in ASGP-R positive HepG2 cells with no significant difference observed in ASGP-R negative HeLa cells. The co-incubation of Gal-APC/DNA complexes with a natural ASGP-R ligand effectively led to a decrease in gene expression, hence providing evidence for the ASGP-R mediated endocytosis of the polyplexes. Importantly, the Gal-APC/DNA complexes induced minimal cytotoxicities in HepG2 cells at the N/P ratios tested. Taken together, the galactose-functionalized cationic polycarbonate diblock copolymer has potential for use as a non-viral gene vector for the targeted delivery of therapeutic genes to hepatocytes in the treatment of liver diseases.

Mechanically tuneable physical nanocomposite hydrogels from polyelectrolyte complex templated silica nanoparticles for anionic therapeutic delivery.

Hydrogels have shown great promise for drug delivery and tissue engineering but can be limited in practical applications by poor mechanical performance. The incorporation of polymer grafted silica nanoparticles as chemical or physical crosslinkers in in situ polymerised nanocomposite hydrogels has been widely researched to enhance their mechanical properties. Despite the enhanced mechanical stiffness, tensile strength, and self-healing properties, there remains a need for the development of simpler and modular approaches to obtain nanocomposite hydrogels. Herein, we report a facile protocol for the polyelectrolyte complex (PEC) templated synthesis of organic-inorganic hybrid poly(ethylenimine) functionalised silica nanoparticles (PEI-SiNPs) and their use as multifunctional electrostatic crosslinkers with hyaluronic acid (HA) to form nanocomposite hydrogels. Upon mixing, electrostatic interactions between cationic PEI-SiNPs and anionic HA resulted in the formation of a coacervate nanocomposite hydrogel with enhanced mechanical stiffness that can be tuned by varying the ratios of PEI-SiNPs and HA present. The reversible electrostatic interactions within the hydrogel networks also enabled self-healing and thixotropic properties. The excess positive charge present within the PEI-SiNPs facilitated high loading and retarded the release of the anionic anti-cancer drug methotrexate from the nanocomposite hydrogel. Furthermore, the electrostatic complexation of PEI-SiNP and HA was found to mitigate haemotoxicity concerns associated with the use of high molecular weight PEI. The method presented herein offers a simpler and more versatile strategy for the fabrication of coacervate nanocomposite hydrogels with tuneable mechanical stiffness and self-healing properties for drug delivery applications.

Novel biodegradable block copolymers of poly(ethylene glycol) (PEG) and cationic polycarbonate: effects of peg configuration on gene delivery.

A novel amine-functionalized polycarbonate was synthesized and its excellent gene transfection ability in vitro is demonstrated. In the framework of adapting the cationic polycarbonate for in vivo gene delivery applications, here the design and synthesis of biodegradable block copolymers of poly(ethylene glycol) (PEG) and amine-functionalized polycarbonate with a well-defined molecular architecture and molecular weight is achieved by metal-free organocatalytic ring-opening polymerization. Copolymers in triblock cationic polycarbonate-block-PEG-block-cationic polycarbonate and diblock PEG-block-cationic polycarbonate configurations, in comparison with a non-PEGylated cationic polycarbonate control, are investigated for their influence on key aspects of gene delivery. Among the polymers with similar molecular weights and N content, the triblock copolymer exhibit more favorable physicochemical (i.e., DNA binding, size, zeta-potential, and in vitro stability) and biological (i.e., cellular uptake and luciferase reporter gene expression) properties. Importantly, the various cationic polycarbonate/DNA complexes are biocompatible, inducing minimal cytotoxicities and hemolysis. These results suggest that the triblock copolymer is a more useful architecture in future cationic polymer designs for successful systemic therapeutic applications.

Polyelectrolyte complex templated synthesis of monodisperse, sub-100 nm porous silica nanoparticles for cancer targeted and stimuli-responsive drug delivery.

Porous silica nanoparticles (PSiNPs) have long attracted interest in drug delivery research. However, conventional synthesis methods for sub-100 nm, functionalised PSiNPs typically give poor monodispersity, reproducibility, or involve complex synthetic protocols. We report a facile, reproducible, and cost-effective one-pot method for the synthesis of cancer targeting and pH responsive PSiNPs in this size range, without the need for post-synthetic modification. This was achieved by using monodisperse l-arginine (Arg)/ poly(acrylic acid) (PAA) polyelectrolyte complexes (PECs) as soft templates for silane hydrolysis and condensation. Highly uniform PSiNPs with tunable size control between 42 and 178 nm and disordered pore structure (1.1-2.7 nm) were obtained. Both PAA and Arg were retained within the PSiNPs, which enabled a high doxorubicin hydrochloride (Dox) loading capacity (22% w/w) and a 4-fold increase in drug release under weakly acidic pH compared to physiological pH. The surface presentation of Arg conferred significantly higher intracellular accumulation of Arg/PAA-PSiNPs in patient-derived glioblastoma cells compared to non-tumorigenic neural progenitor cells, which effectively translated to lower IC50 values for Dox-loaded Arg/PAA-PSiNPs than non-functionalised PSiNPs. This work brings forward new insights for the development of monodisperse PSiNPs with highly desirable built-in functionalities for biomedical applications.

Antitumor activity of natural compounds, curcumin and PKF118-310, as Wnt/ß-catenin antagonists against human osteosarcoma cells.

Aberrant activation of the Wnt/ß-catenin signaling pathway promotes osteosarcoma tumorigenesis and metastasis. In this study, we tested the hypothesis that osteosarcoma progression may be delayed by disrupting the Wnt/ß-catenin pathway using small molecule inhibitors such as curcumin and PKF118-310. Effective inhibitions of the Wnt/ß-catenin pathway by curcumin and PKF118-310 in osteosarcoma cells were shown by the suppression of both intrinsic and activated ß-catenin/Tcf transcriptional activities using luciferase reporter assays. Western blot analysis revealed that there was no change in the amount of cytosolic ß-catenin, although nuclear ß-catenin was markedly reduced by treatment with either compounds. We next performed wound healing and Matrigel invasion assays and observed a dose-dependent decrease in osteosarcoma cell migration and invasion with curcumin and PKF118-310 treatment. Overexpression of the wild-type ß-catenin plasmid in osteosarcoma cells resulted in enhanced cell invasiveness but this effect was significantly overcome by curcumin. Gelatin zymography and Western blotting showed that reduced cell invasion with curcumin and PKF118-310 treatment correlated with the activity and protein level of matrix metalloproteinase-9 under conditions of intrinsic or extrinsic Wnt/ß-catenin activation. Using cell apoptosis assay and cell cycle analysis, we further showed that the anti-proliferative effect of PKF118-310 is attributed to PKF118-310-induced apoptosis and G2/M phase arrest. Lastly, we observed that these anti-cancer effects correlated with the decreased expression of cyclin D1, c-Myc and survivin. Our findings strongly suggest that curcumin and PKF118-310 have great therapeutic potential for the treatment of osteosarcoma.

Nanoparticle-Loaded Hydrogel for the Light-Activated Release and Photothermal Enhancement of Antimicrobial Peptides.

Rising concerns over multidrug-resistant bacteria have necessitated an expansion to the current antimicrobial arsenal and forced the development of novel delivery strategies that enhance the efficacy of existing treatments. Antimicrobial peptides (AMPs) are a promising antibiotic alternative that physically disrupts the membrane of bacteria, resulting in rapid bactericidal activity; however, clinical translation of AMPs has been hindered by their susceptibility to protease degradation. Through the co-loading of liposomes encapsulating model AMP, IRIKIRIK-CONH2 (IK8), and gold nanorods (AuNRs) into a poly(ethylene glycol) (PEG) hydrogel, we have demonstrated the ability to protect encapsulated materials from proteolysis and provide the first instance of the triggered AMP release. Laser irradiation at 860 nm, at 2.1 W cm-2, for 10 min led to the photothermal triggered release of IK8, resulting in bactericidal activity against Gram-negative Pseudonomas aeruginosa and Gram-positive Staphylococcus aureus. Furthermore, by increasing the laser intensity to 2.4 W cm-2, we have shown the thermal enhancement of AMP activity. The photothermal triggered release, and enhancement of AMP efficacy, was demonstrated to treat two rounds of fresh S. aureus, indicating that the therapeutic gel has the potential for multiple rounds of treatment. Taken together, this novel therapeutic hydrogel system demonstrates the stimuli-responsive release of AMPs with photothermal enhanced antimicrobial efficacy to treat pathogenic bacteria.

Efficient delivery of Bcl-2-targeted siRNA using cationic polymer nanoparticles: downregulating mRNA expression level and sensitizing cancer cells to anticancer drug.

In this study, cationic nanoparticles self-assembled from the amphiphilic copolymer poly(N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate) (P(MDS-co-CES) were synthesized and used to deliver Bcl-2 targeted siRNA into HepG2, HeLa and MDA-MB-231 cell lines, and downregulate Bcl-2 mRNA expression levels. Confocal microscopic studies show that the nanoparticles were able to complex with siRNA and deliver it inside the cells efficiently, but siRNA was easily dissociated from the complexes in the cytoplasm for its biological functions. Bcl-2 mRNA expression levels as low as 10% were achieved after treatment with nanoparticle/siRNA complexes. The downregulation efficiency of Bcl-2 mRNA level was similar to that mediated by Lipofectamine but higher than that induced by PEI. PEG was also conjugated to siRNA via a cleavable disulfide bond, and nanoparticle/siRNA-PEG complexes showed no significant protein adsorption as compared with 26 and 17% for blank nanoparticles and nanoparticle/siRNA complexes, respectively. The presence of serum caused slight aggregation of nanoparticle/siRNA or nanoparticle/siRNA-PEG complexes. However, the size of the complexes was still below 250 nm after being incubated in PBS containing 10% serum for 4 h. On the other hand, PEGylated siRNA delivered by the nanoparticles downregulated Bcl-2 mRNA expression level in the cells as efficiently as unmodified siRNA. Bcl-2 protein was also downregulated efficiently by nanoparticle/siRNA complexes in all cell lines tested. The downregulation of Bcl-2 mRNA or Bcl-2 protein did not show significant cell death in the tested siRNA and polymer concentration range. However, the delivery of siRNA sensitized HeLa cells to paclitaxel treatment, yielding significant improvement over the untreated cells (p<0.05). These cationic nanoparticles may be potentially employed to downregulate Bcl-2 expression and sensitize cancer cells to anticancer drugs for more efficient chemotherapy.

Graphene oxide exhibits differential mechanistic action towards Gram-positive and Gram-negative bacteria.

The antibacterial nature of graphene oxide (GO) has stimulated wide interest in the medical field. Although the antibacterial activity of GO towards bacteria has been well studied, a deeper understanding of the mechanism of action of GO is still lacking. The objective of the study was to elucidate the difference in the interactions of GO towards Gram-positive and Gram-negative bacteria. The synthesized GO was characterized by Ultraviolet-visible spectroscopy (UV-vis), Raman and Attenuated Total Reflectance-Fourier-transform infrared spectroscopy (ATR-FTIR). Viability, time-kill and Lactose Dehydrogenase (LDH) release assays were carried out along with FESEM, TEM and ATR-FTIR analysis of GO treated bacterial cells. Characterizations of synthesized GO confirmed the transition of graphene to GO and the antibacterial activity of GO was concentration and time-dependent. Loss of membrane integrity in bacteria was enhanced with increasing GO concentrations and this corresponded to the elevated release of LDH in the reaction medium. Surface morphology of GO treated bacterial culture showed apparent differences in the mechanism of action of GO towards Gram-positive and Gram-negative bacteria where cell entrapment was mainly observed for Gram-positive Staphylococcus aureus and Enterococcus faecalis whereas membrane disruption due to physical contact was noted for Gram-negative Escherichia coli and Pseudomonas aeruginosa. ATR-FTIR characterizations of the GO treated bacterial cells showed changes in the fatty acids, amide I and amide II of proteins, peptides and amino acid regions compared to untreated bacterial cells. Therefore, the data generated further enhance our understanding of the antibacterial activity of GO towards bacteria.

Stimuli-Responsive Release of Antimicrobials Using Hybrid Inorganic Nanoparticle-Associated Drug-Delivery Systems.

Recently, the combination of metallic nanoparticles (NPs) of Au, Ag, Fe2 O3 , and Fe3 O4 with traditional soft matter drug-delivery systems has emerged as a promising strategy to achieve site-specific and controlled release of antimicrobial agents. By harnessing the plasmonic and magnetic properties of inorganic NPs, the disruption of antibiotic-loaded liposomes, polymersomes, and hydrogels can be remotely triggered by mechanisms such as photo- and magneto-thermal effects, microbubble cavitation, magnetic positioning, and pH-changes, hence offering significant advantages in improving antibacterial efficacy, reducing side effects, and in overcoming antimicrobial resistance. This review highlights the latest development of stimuli-responsive antibiotic delivery systems incorporating inorganic NPs. The methods employed for preparation of hybrid inorganic NP-associated drug-delivery systems and the effects this has upon the system are discussed. Finally, a detailed exposition of the NP-mediated triggering mechanisms is provided and pertinent examples of their use in antimicrobial applications are presented.

Recommendations for clinical translation of nanoparticle-enhanced radiotherapy.

A multi-disciplinary cooperative for nanoparticle-enhanced radiotherapy (NERT) has been formed to review the current status of the field and identify key stages towards translation. Supported by the Colorectal Cancer Healthcare Technologies Cooperative, the cooperative comprises a diverse cohort of key contributors along the translation pathway including academics of physics, cancer and radio-biology, chemistry, nanotechnology and clinical trials, clinicians, manufacturers, industry, standards laboratories, policy makers and patients. Our aim was to leverage our combined expertise to devise solutions towards a roadmap for translation and commercialisation of NERT, in order to focus research in the direction of clinical implementation, and streamline the critical pathway from basic science to the clinic. A recent meeting of the group identified barriers to and strategies for accelerated clinical translation. This commentary reports the cooperative's recommendations. Particular emphasis was given to more standardised and cohesive research methods, models and outputs, and reprioritised research drivers including patient quality of life following treatment. Nanoparticle design criteria were outlined to incorporate scalability of manufacture, understanding and optimisation of biological mechanisms of enhancement and in vivo fate of nanoparticles, as well as existing design criteria for physical and chemical enhancement. In addition, the group aims to establish a long-term and widespread international community to disseminate key findings and create a much-needed cohesive body of evidence necessary for commercial and clinical translation.

Short Synthetic α-Helical-Forming Peptide Amphiphiles for Fungal Keratitis Treatment In Vivo.

The emergence of fungal keratitis is on the rise globally. However, current antifungal therapeutics are ineffective in severe keratomycosis. Previously reported α-helical peptides comprising 8-14 amino acids demonstrate broad-spectrum antimicrobial activity both in vitro and in vivo. Here, α-helical peptides of the optimized sequences are investigated for antifungal biofilm in vitro and in vivo using a fungal biofilm-caused mouse keratitis model. The peptides with the optimal composition demonstrate higher α-helical propensity and improve antifungal activity in dispersing Candida albicans biofilm in vitro. Moreover, the optimized α-helical peptides are not only effective in treating C. albicans biofilm-induced keratitis in mice, they are also nontoxic to the mice eyes. These peptides have the potential to be developed as antifungal agents for the treatment of C. albicans biofilm-caused keratitis.

Biodegradable cationic poly(carbonates): Effect of varying side chain hydrophobicity on key aspects of gene transfection.

The degree of hydrophobicity in cationic polymers plays an important but often underappreciated role in the safety and efficacy of gene delivery processes. In order to further elucidate structure-activity relationships of biodegradable cationic poly(carbonate) gene carriers, we synthesized a series of narrowly dispersed homo-polymers via metal-free organocatalytic living ring-opening polymerization (ROP) of cyclic carbonate monomers bearing either alkyl (propyl, hexyl or nonyl) or 4-methyl benzyl halide side chains. The polymers were then quaternized using bis-tertiary amines to install both quaternary ammoniums and tertiary amines for DNA binding and endosomal escape, respectively. Among the polymers with similar molecular lengths and charge densities, it was found that an increase in side chain alkyl spacer length from 3 to 6 carbons significantly enhanced cellular uptake and luciferase gene expression in HepG2 and HeLa cell lines without causing overt hemolysis and cytotoxicity. A further increase of side chain alkyl length to 9 carbons, however, led to a drastic decline in gene expression due to increased cellular toxicity, which was correlated with an increased disruption and lysis of red blood cell membranes. Interestingly, the incorporation of an aromatic 4-methyl benzyl spacer increased DNA binding strength, reduced particle sizes of resultant DNA complexes, and enhanced cellular uptake, leading to improved luciferase gene expression, albeit with higher levels of hemolysis and cytotoxicity. Taken together, the findings of this study demonstrate that a delicate balance between cationic charge density and hydrophobicity could be achieved by utilizing a hexyl spacer in the side chains of cationic poly(carbonates), hence providing insights on the future development of non-viral cationic polymeric gene delivery systems. STATEMENT OF SIGNIFICANCE: Owing to their ease of synthesis and well-controlled polymerization, biodegradable cationic poly(carbonates) have emerged as a highly promising class of biomaterials for gene delivery. The hydrophobicity of side chains in cationic polymers plays an important but often underappreciated role in influencing key aspects of gene transfection. In our efforts to improve gene transfection and understand structure-activity relationships, we synthesized a series of cationic polymers bearing a common poly(carbonate) backbone, and with side chains containing various hydrophobic spacers (propyl, hexyl, 4-methyl benzyl or nonyl) before the cationic moiety. A moderate degree of hydrophobicity was optimal as the cationic poly(carbonate) with hexyl side chains mediated high gene transfection efficiencies while causing low cytotoxicities.

Multibranched Gold Nanoparticles with Intrinsic LAT-1 Targeting Capabilities for Selective Photothermal Therapy of Breast Cancer.

Because of the critical role of the large neutral amino acid transporter-1 (LAT-1) in promoting tumor growth and proliferation, it is fast emerging as a highly attractive biomarker for the imaging and treatment of human malignancies, including breast cancer. While multibranched gold nanoparticles (AuNPs) have emerged as a promising modality in the photothermal therapy (PTT) of cancers, some of the key challenges limiting their clinical translation lie in the need to develop reproducible and cost-effective synthetic methods as well as the selective accumulation of sufficient AuNPs at tumor sites. In this study, we report a simple and direct seed-mediated synthesis of monodispersed multibranched AuNPs using the catechol-containing LAT-1 ligands, L- and D-dopa, to confer active cancer targeting. This route obviates the need for additional conjugation with targeting moieties such as peptides or antibodies. Nanoflower-like AuNPs (AuNF) with diameters of approximately 46, 70, and 90 nm were obtained and were found to possess excellent colloidal stability and biocompatibility. A significantly higher intracellular accumulation of the L- and D-dopa functionalized AuNFs was observed in a panel of breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468, and MDA-MB-453) when compared to the nontargeting control AuNFs synthesized with dopamine and 4-ethylcatechol. Importantly, no significant difference in uptake between the targeting and nontargeting AuNFs was observed in a non-tumorigenic MCF-10A breast epithelial cell line, hence demonstrating tumor selectivity. For PTT of breast cancer, Ag+ was introduced during synthesis to obtain L-dopa functionalized nanourchin-like AuNPs (AuNUs) with strong near-infrared (NIR) absorbance. The L-dopa functionalized AuNUs mediated selective photothermal ablation of the triple negative MDA-MB-231 breast cancer cell line and sensitized the cells to the anticancer drugs cisplatin and docetaxel. This work brings forward an effective strategy for the facile preparation of cancer targeting multibranched AuNPs with potential for the in vivo PTT of breast cancer.