Jayanta Bhattacharyya

Self-Assembly of Thermally Responsive Nanoparticles of a Genetically Encoded Peptide Polymer by Drug Conjugation†

Chimeric polypeptides (CPs) that are derived from elastin-like polypeptides (ELPs) can self-assemble to form nanoparticles by site-specific covalent attachment of hydrophobic molecules to one end of the biopolymer backbone. Molecules with a distribution coefficient greater than 1.5 impart sufficient amphiphilicity to drive self-assembly into sub-100 nm nanoparticles.

A paclitaxel-loaded recombinant polypeptide nanoparticle outperforms Abraxane in multiple murine cancer models

Packaging clinically relevant hydrophobic drugs into a self-assembled nanoparticle can improve their aqueous solubility, plasma half-life, tumor specific uptake and therapeutic potential. To this end, here we conjugated paclitaxel (PTX) to recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into ~60-nm diameter near-monodisperse nanoparticles that increased the systemic exposure of PTX by 7-fold compared to free drug and 2-fold compared to the FDA approved taxane nanoformulation (Abraxane®). The tumor uptake of the CP-PTX nanoparticle was 5-fold greater than free drug and 2-fold greater than Abraxane. In a murine cancer model of human triple negative breast cancer and prostate cancer, CP-PTX induced near complete tumor regression after a single dose in both tumor models, whereas at the same dose, no mice treated with Abraxane survived for more than 80 days (breast) and 60 days (prostate) respectively. These results show that a molecularly engineered nanoparticle with precisely engineered design features outperforms Abraxane, the current gold standard for paclitaxel delivery.

Ring-Opening Polymerization of Prodrugs: A Versatile Approach to Prepare Well-Defined Drug-Loaded Nanoparticles†

The synthesis of polymer-drug conjugates from prodrug monomers consisting of a cyclic polymerizable group that is appended to a drug through a cleavable linker is achieved by organocatalyzed ring-opening polymerization. The monomers polymerize into well-defined polymer prodrugs that are designed to self-assemble into nanoparticles and release the drug in response to a physiologically relevant stimulus. This method is compatible with structurally diverse drugs and allows different drugs to be copolymerized with quantitative conversion of the monomers. The drug loading can be controlled by adjusting the monomer(s)/initiator feed ratio and drug release can be encoded into the polymer by the choice of linker. Initiating these monomers from a poly(ethylene glycol) macroinitiator results in amphiphilic diblock copolymers that spontaneously self-assemble into micelles with a long plasma circulation, which is useful for systemic therapy.

A Noncanonical Function of Sortase Enables Site‐Specific Conjugation of Small Molecules to Lysine Residues in Proteins

We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full-length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε-amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site-specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site-specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox.

iTEP Nanoparticle-Delivered Salinomycin Displays an Enhanced Toxicity to Cancer Stem Cells in Orthotopic Breast Tumors

Salinomycin (Sali) has selective toxicity to cancer stem cells (CSCs), a subpopulation of cancer cells that have been recently linked with tumor multidrug resistance (MDR). To utilize its selective toxicity for cancer therapy, we sought to devise a nanoparticle (NP) carrier to deliver Sali to solid tumors through the enhanced permeability and retention effect and, hence, to increase its exposure to CSCs. First, hydrophobic Sali was conjugated to a hydrophilic, immune-tolerant, elastin-like polypeptide (iTEP); the amphiphilic iTEP–Sali conjugates self-assemble into NPs. Next, free Sali was encapsulated into the NPs alone or with two additives, N,N-dimethylhexylamine (DMHA) and α-tocopherol. The coencapsulation significantly improved the loading efficiency and release profile of Sali. The resulting NPs of the coencapsulation, termed as iTEP–Sali NP3s, have an in vitro release half-life of 4.1 h, four times longer than iTEP–Sali NP2s, the NPs that have encapsulated Sali only. Further, the NP3 formulation increases the plasma area under curve and the tumor accumulation of Sali by 10 and 2.4 times, respectively. Lastly, these improved pharmacokinetic and tumor accumulation profiles are consistent with a boost of CSC-elimination effect of Sali in vivo. In NP3-treated 4T1 orthotopic tumors, the mean CSC frequency is 55.62%, a significant reduction from the mean frequencies of untreated tumors, 75.00%, or free Sali-treated tumors, 64.32%. The CSC-elimination effect of the NP3 can further translate to a delay of tumor growth. Given the role of CSCs in driving tumor MDR and recurrence, it could be a promising strategy to add the NP3 to conventional cancer chemotherapies to prevent or reverse the MDR.

A Genetically Engineered Thermally Responsive Sustained Release Curcumin Depot to Treat Neuroinflammation

Radiculopathy, a painful neuroinflammation that can accompany intervertebral disc herniation, is associated with locally increased levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Systemic administration of TNF antagonists for radiculopathy in the clinic has shown mixed results, and there is growing interest in the local delivery of anti-inflammatory drugs to treat this pathology as well as similar inflammatory events of peripheral nerve injury. Curcumin, a known antagonist of TNFα in multiple cell types and tissues, was chemically modified and conjugated to a thermally responsive elastin-like polypeptide (ELP) to create an injectable depot for sustained, local delivery of curcumin to treat neuroinflammation. ELPs are biopolymers capable of thermally-triggered in situ depot formation that have been successfully employed as drug carriers and biomaterials in several applications. ELP-curcumin conjugates were shown to display high drug loading, rapidly release curcumin in vitro via degradable carbamate bonds, and retain in vitro bioactivity against TNFα-induced cytotoxicity and monocyte activation with IC50 only two-fold higher than curcumin. When injected proximal to the sciatic nerve in mice via intramuscular (i.m.) injection, ELP-curcumin conjugates underwent a thermally triggered soluble-insoluble phase transition, leading to in situ formation of a depot that released curcumin over 4days post-injection and decreased plasma AUC 7-fold.

Covalent Grafting of Common Trihydroxymethylaminomethane in the Headgroup Region Imparts High Serum Compatibility and Mouse Lung Transfection Property to Cationic Amphiphile

Clinical success of cationic transfection lipids in nonviral gene therapy continues to remain critically dependent on the use of serum compatible cationic amphiphiles efficient in delivering genes into our body cells. To this end, we demonstrate that covalent grafting of simple Tris-base component of the widely used biological Tris buffer in the headgroup region is capable of imparting high serum compatibility and intravenous mouse lung transfection properties to cationic amphiphile.

A quantitative study of the intracellular fate of pH-responsive doxorubicin-polypeptide nanoparticles

Abstract Nanoscale carriers with an acid‐labile linker between the carrier and drug are commonly used for drug delivery. However, their efficacy is potentially limited by inefficient linker cleavage, and lysosomal entrapment of drugs. To address these critical issues, we developed a new imaging method that spatially overlays the location of a nanoparticle and the released drug from the nanoparticle, on a map of the local intracellular pH that delineates individual endosomes and lysosomes, and the therapeutic intracellular target of the drug—the nucleus. We used this method to quantitatively map the intracellular fate of micelles of a recombinant polypeptide conjugated with doxorubicin via an acid‐labile hydrazone linker as a function of local pH and time within live cells. We found that hydrolysis of the acid‐labile linker is incomplete because the pH range of 4–7 in the endosomes and lysosomes does not provide complete cleavage of the drug from the nanoparticle, but that once cleaved, the drug escapes the acidic endo‐lysosomal compartment into the cytosol and traffics to its therapeutic destination—the nucleus. This study also demonstrated that unlike free drug, which enters the cytosol directly through the cell membrane and then traffics into the nucleus, the nanoparticle‐loaded drug almost exclusively traffics into endosomes and lysosomes upon intracellular uptake, and only reaches the nucleus after acid‐triggered drug release in the endo‐lysosomes. This methodology provides a better and more quantitative understanding of the intracellular behavior of drug‐loaded nanoparticles, and provides insights for the design of the next‐generation of nanoscale drug delivery systems. Graphical Abstract Figure. No Caption available.

Single subcutaneous administration of RGDK-lipopeptide: rhPDGF-B gene complex heals wounds in streptozotocin-induced diabetic rats

Development of effective therapeutics for chronic wounds remains a formidable clinical challenge. Deficiency of growth factors is of paramount importance among the multitude of factors contributing to the pathogenesis of diabetic wounds. Clinical interest has been witnessed in the past for exogenous applications of platelet derived growth factor B (PDGF-B) in chronic nonhealing wounds. However, accomplishing even modest favorable clinical effects in such topical applications requires large and repeated doses of PDGF-B proteins. Chronic wounds are being increasingly circumvented by gene therapy approach and to this end, cationic liposomes are emerging as promising nonviral carriers for delivering various growth factors encoding therapeutic genes to wound beds. However, as in case of topical application of growth factors, all the prior studies on the use of cationic liposomes in nonviral gene therapy of wounds involved repeated injections of cationic liposome:cDNA complexes over several weeks for ensuring complete wound healing. Herein, we show that a single subcutaneous administration of an electrostatic complex of rhPDGF-B plasmid, integrin receptor selective RGDK-lipopeptide 1 and cholesterol (as auxiliary lipid) is capable of healing wounds in streptozotocin-induced diabetic Sprague-Dawley rats (as model of chronic wounds). Western blot analysis revealed significant expression of rhPDGF-B in mouse fibroblast cells transfected with RGDK-lipopeptide 1:rhPDGF-B lipoplex. The transfection efficiencies of the RGDK-lipopeptide 1 in mouse and human fibroblast cells preincubated with various monoclonal anti-integrin receptor antibodies support the notion that the cellular uptake of the RGDK-lipopeptide 1:DNA complexes in fibroblast cells is likely to be selectively mediated by alpha5beta1 integrin receptors. Findings in the histopathological stainings using both hematoxylin and eosin (H & E) as well as Massons Trichrome staining revealed a significantly higher degree of epithelization, keratization, fibrocollagenation and blood vessel formation in rats treated with RGDK-lipopeptide 1:rhPDGF compared to those in rats treated with vehicle alone.

Encapsulating a Hydrophilic Chemotherapeutic into Rod-Like Nanoparticles of a Genetically Encoded Asymmetric Triblock Polypeptide Improves Its Efficacy

Encapsulating hydrophilic chemotherapeutics into the core of polymeric nanoparticles can improve their therapeutic efficacy by increasing their plasma half-life, tumor accumulation and intracellular uptake, and by protecting them from premature degradation. To achieve these goals, we designed a recombinant asymmetric triblock polypeptide (ATBP) that self-assembles into rod-shaped nanoparticles, and which can be used to conjugate diverse hydrophilic molecules, including chemotherapeutics, into their core. These ATBPs consist of three segments: a biodegradable elastin-like polypeptide, a hydrophobic Tyrosine-rich segment, and a short Cysteine-rich segment, that spontaneously self-assemble into rod-shaped micelles. Covalent conjugation of a structurally diverse set of hydrophilic small molecules, including a hydrophilic chemotherapeutic -gemcitabine- to the Cysteine residues also leads to formation of nanoparticles over a range of ATBP concentrations. Gemcitabine-loaded ATBP nanoparticles have significantly better tumor regression compared to free drug in a murine cancer model. This simple strategy of encapsulation of hydrophilic small molecules by conjugation to an ATBP can be used to effectively deliver a range of water-soluble drugs and imaging agents in vivo.