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Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...

Stanford University

Layer-by-layer nanoparticles (NPs) are modular drug delivery vehicles that incorporate multiple functional materials through sequential deposition of polyelectrolytes onto charged nanoparticle cores. Herein, we combined the multicomponent features and tumor targeting capabilities of layer-by-layer assembly with functional biosensing peptides to create a new class of nanotheranostics. These NPs encapsulate a high weight percentage of siRNA while also carrying a synthetic biosensing peptide on the surface that is cleaved into a urinary reporter upon exposure to specific proteases overexpressed in the tumor microenvironment. Importantly, this biosensor reports back on a molecular signature characteristic to metastatic tumors and associated with poor prognosis, MMP9 protease overexpression. This nanotheranostic mediates noninvasive urinary-based diagnostics in mouse models of three different cancers with simultaneous gene silencing in flank and metastatic mouse models of ovarian cancer.

Theranostic Layer-by-Layer Nanoparticles for Simultaneous Tumor Detection and Gene Silencing.

Using siRNA therapeutics to treat hematologic malignancies has been unsuccessful because blood cancer cells exhibit remarkable resistance to standard transfection methods. Herein we report the successful delivery of siRNA therapeutics with a dual-targeted, layer-by-layer nanoparticle (LbL-NP). The LbL-NP protects siRNA from nucleases in the bloodstream by embedding it within polyelectrolyte layers that coat a polymeric core. The outermost layer consists of hyaluronic acid (a CD44-ligand) covalently conjugated to CD20 antibodies. The CD20/CD44 dual-targeting outer layer provides precise binding to blood cancer cells, followed by receptor-mediated endocytosis of the LbL-NP. We use this siRNA delivery platform to silence B-cell lymphoma 2 (BCL-2), a pro-survival protein,  and . The dual-targeting approach significantly enhanced internalization of BCL-2 siRNA in lymphoma and leukemia cells, which led to significant downregulation of BCL-2 expression. Systemic administration of the dual-targeted, siRNA-loaded nanoparticle induced apoptosis and hampered proliferation of blood cancer cells both in cell culture and in orthotopic non-Hodgkin's lymphoma animal models. These results provide the basis for approaches to targeting blood-borne cancers and other diseases, and suggest that LbL nanoassemblies are a promising approach for delivering therapeutic siRNA to hematopoetic cell types that are known to evade transfection by other means.

Binary Targeting of siRNA to Hematologic Cancer Cells  using Layer-by-Layer Nanoparticles.

Treatment of patients with diabetes with insulin and pramlintide (an amylin analogue) is more effective than treatment with insulin only. However, because mixtures of insulin and pramlintide are unstable and have to be injected separately, amylin analogues are only used by 1.5% of people with diabetes needing rapid-acting insulin. Here, we show that the supramolecular modification of insulin and pramlintide with cucurbit[7]uril-conjugated polyethylene glycol improves the pharmacokinetics of the dual-hormone therapy and enhances postprandial glucagon suppression in diabetic pigs. The co-formulation is stable for over 100 h at 37 °C under continuous agitation, whereas commercial formulations of insulin analogues aggregate after 10 h under similar conditions. In diabetic rats, the administration of the stabilized co-formulation increased the area-of-overlap ratio of the pharmacokinetic curves of pramlintide and insulin from 0.4 ± 0.2 to 0.7 ± 0.1 (mean ± s.d.) for the separate administration of the hormones. The co-administration of supramolecularly stabilized insulin and pramlintide better mimics the endogenous kinetics of co-secreted insulin and amylin, and holds promise as a dual-hormone replacement therapy.

A co-formulation of supramolecularly stabilized insulin and pramlintide enhances mealtime glucagon suppression in diabetic pigs.

Insulin has been used to treat diabetes for almost 100 years; yet, current rapid-acting insulin formulations do not have sufficiently fast pharmacokinetics to maintain tight glycemic control at mealtimes. Dissociation of the insulin hexamer, the primary association state of insulin in rapid-acting formulations, is the rate-limiting step that leads to delayed onset and extended duration of action. A formulation of insulin monomers would more closely mimic endogenous postprandial insulin secretion, but monomeric insulin is unstable in solution using present formulation strategies and rapidly aggregates into amyloid fibrils. Here, we implement high-throughput-controlled radical polymerization techniques to generate a large library of acrylamide carrier/dopant copolymer (AC/DC) excipients designed to reduce insulin aggregation. Our top-performing AC/DC excipient candidate enabled the development of an ultrafast-absorbing insulin lispro (UFAL) formulation, which remains stable under stressed aging conditions for 25 ± 1 hours compared to 5 ± 2 hours for commercial fast-acting insulin lispro formulations (Humalog). In a porcine model of insulin-deficient diabetes, UFAL exhibited peak action at 9 ± 4 min, whereas commercial Humalog exhibited peak action at 25 ± 10 min. These ultrafast kinetics make UFAL a promising candidate for improving glucose control and reducing burden for patients with diabetes.

An ultrafast insulin formulation enabled by high-throughput screening of engineered polymeric excipients.

Cancer immunotherapy can be augmented with toll-like receptor agonist (TLRa) adjuvants, which interact with immune cells to elicit potent immune activation. Despite their potential, use of many TLRa compounds has been limited clinically due to their extreme potency and lack of pharmacokinetic control, causing systemic toxicity from unregulated systemic cytokine release. Herein, we overcome these shortcomings by generating poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) nanoparticles (NPs) presenting potent TLR7/8a moieties on their surface. The NP platform allows precise control of TLR7/8a valency and resulting surface presentation through self-assembly using nanoprecipitation. We hypothesize that the pharmacokinetic profile of the NPs minimizes systemic toxicity, localizing TLR7/8a presentation to the tumor bed and tumor-draining lymph nodes. In conjunction with antiprogrammed death-ligand 1 (anti-PD-L1) checkpoint blockade, peritumoral injection of TLR7/8a NPs slows tumor growth, extends survival, and decreases systemic toxicity in comparison to the free TLR7/8a in a murine colon adenocarcinoma model. These NPs constitute a modular platform for controlling pharmacokinetics of immunostimulatory molecules, resulting in increased potency and decreased toxicity.

Nanoparticles Presenting Potent TLR7/8 Agonists Enhance Anti-PD-L1 Immunotherapy in Cancer Treatment.

The oncogenic transcription factor STAT3 is aberrantly activated in 70% of breast cancers, including nearly all triple-negative breast cancers (TNBC). Since STAT3 is difficult to target directly, we considered whether metabolic changes driven by activated STAT3 could provide a therapeutic opportunity. We found that STAT3 prominently modulated several lipid classes, with most profound effects on N-acyl taurine and arachidonic acid, both of which are involved in plasma membrane remodeling. To exploit these metabolic changes therapeutically, we screened a library of layer-by-layer (LbL) nanoparticles (NP) differing in the surface layer that modulates interactivity with the cell membrane. We found that poly-L-glutamic acid (PLE)-coated NPs bind to STAT3-transformed breast cancer cells with 50% greater efficiency than to non-transformed cells, and the heightened PLE-NP binding to TNBC cells was attenuated by STAT3 inhibition. This effect was also observed in densely packed three-dimensional breast cancer organoids. As STAT3-transformed cells show greater resistance to cytotoxic agents, we evaluated if enhanced targeted delivery via PLE-NPs would provide a therapeutic advantage. We found that cisplatin-loaded PLE-NPs induced apoptosis of STAT3-driven cells at lower doses compared to both unencapsulated cisplatin and cisplatin-loaded non-targeted NPs. In addition, since radiation is commonly used in breast cancer treatment, and may alter cellular lipid distribution, we analyzed its effect on PLE-NP-cell binding. Irradiation of cells enhanced the STAT3-targeting properties of PLE-NPs in a dose-dependent manner, suggesting potential synergies between these therapeutic modalities. These findings suggest that cellular lipid changes driven by activated STAT3 may be exploited therapeutically using unique LbL nanoparticles.

Santiago Correa

Department of Materials Science & Engineering

Stanford University

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

Santiago Correa

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Materials Science & Engineering

Stanford University

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

Department of Materials Science & Engineering