Vaginal instillation of small-interfering RNA (siRNA) using liposomes has led to silencing of endogenous genes in the genital tract and protected against challenge from infectious disease. Although siRNA lipoplexes are easily formulated, several of the most effective transfection agents available commercially may be toxic to the mucosal epithelia and none are able to provide controlled or sustained release. Here, we demonstrate an alternate approach, using nanoparticles composed entirely of FDA-approved materials. To render these materials effective for gene silencing we developed novel approaches to load them with high amounts of siRNA. A single dose of siRNA-loaded nanoparticles to the mouse female reproductive tract caused efficient and sustained gene silencing. Knockdown of gene expression was observed proximal (in the vaginal lumen) and distal (in the uterine horns) to the site of topical delivery. In addition, nanoparticles penetrated deep into the epithelial tissue. This is the first report demonstrating that biodegradable polymer nanoparticles are effective delivery vehicles for siRNA in the vaginal mucosa.

PREFACE The primary goal of nanomedicine is to improve clinical outcomes. Toward this end, targeted nanoparticles are engineered to reduce non-productive distribution while improving diagnostic and therapeutic efficacy. Paradoxically, as this field has matured, the notion of ‘targeting’ has been minimized to the concept of increasing affinity of a nanoparticle for its target. This review outlines a holistic view of nanoparticle targeting, in which nanoparticle route of administration, molecular characteristics, and temporal control are potential design variables that must be considered simultaneously. This comprehensive vision for nanoparticle targeting will hasten the integration of nanomedicines into clinical practice.

A key attribute for nanoparticles (NPs) that are used in medicine is the ability to avoid rapid uptake by phagocytic cells in the liver and other tissues. Poly(ethylene glycol) (PEG) coatings has been the gold standard in this regard for several decades. Here, we examined hyperbranched polyglycerols (HPG) as an alternate coating on NPs. In earlier work, HPG was modified with amines and subsequently conjugated to poly(lactic acid) (PLA), but that approach compromised the ability of HPG to resist non-specific adsorptions of biomolecules. Instead, we synthesized a copolymer of PLA-HPG by a one-step esterification. NPs were produced from a single emulsion using PLA-HPG: fluorescent dye or the anti-tumor agent camptothecin (CPT) were encapsulated at high efficiency in the NPs. PLA-HPG NPs were quantitatively compared to PLA-PEG NPs, produced using approaches that have been extensively optimized for drug delivery in humans. Despite being similar in size, drug release profile and in vitro cytotoxicity, the PLA-HPG NPs showed significantly longer blood circulation and significantly less liver accumulation than PLA-PEG. CPT-loaded PLA-HPG NPs showed higher stability in suspension and better therapeutic effectiveness against tumors in vivo than CPT-loaded PLA-PEG NPs. Our results suggest that HPG is superior to PEG as a surface coating for NPs in drug delivery.

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Direct delivery of chemotherapy agents to the brain via degradable polymer delivery systems—such as Gliadel®—is a clinically proven method for treatment of glioblastoma multiforme, but there are important limitations with the current technology—including the requirement for surgery, profound local tissue toxicity, and limitations in diffusional penetration of agents—that limit its application and effectiveness. Here, we demonstrate another technique for direct, controlled delivery of chemotherapy to the brain that provides therapeutic benefit with fewer limitations. In our new approach, camptothecin (CPT)-loaded poly(lacticco-glycolic acid) (PLGA) nanoparticles are infused via convection-enhanced delivery (CED) to a stereotactically defined location in the brain, allowing simultaneous control of location, spread, and duration of drug release. To test this approach, CPT-PLGA nanoparticles (~100 nm in diameter) were synthesized with 25% drug loading. When these nanoparticles were incubated in culture with 9L gliosarcoma cells, the IC50 of CPT-PLGA nanoparticles was 0.04 µM, compared to 0.3 µM for CPT alone. CPT-PLGA nanoparticles stereotactically delivered by CED improved survival in rats with intracranial 9L tumors: the median survival for rats treated with CPT-PLGA nanoparticles (22 days) was significantly longer than unloaded nanoparticles (15 days) and free CPT infusion (17 days). CPT-PLGA nanoparticle treatment also produced significantly more long-term survivors (30% of animals were free of disease at 60 days) than any other treatment. CPT was present in tissues harvested up to 53 days post-infusion, indicating prolonged residence at the local site of administration. These are the first results to demonstrate the effectiveness of combining polymer-controlled release nanoparticles with CED in treating fatal intracranial tumors.

Encapsulation of tumor-associated antigens (TAA) in polymer nanoparticles is a promising approach to increasing the efficiency of antigen (Ag) delivery for antitumor vaccines. We optimized a polymer preparation method to deliver both defined tumor-associated proteins and the complex mixtures of tumor Ags present in tumors. Tumor Ags were encapsulated in a biodegradable, 50:50 poly(D,L-lactide co-glycolide) copolymer (PLGA) by emulsification and solvent extraction. Two particular Ags were studied, gp100 (a melanoma-associated antigen) and ovalbumin (OVA), as well as mixtures of proteins and lysates of tumor cells. The efficiency of encapsulation was measured by protein assays of dissolved nanoparticles. Ag stability after release from nanoparticles was verified by SDS-acrylamide gel electrophoresis and Western blot analysis. Molecular weight and protein loading interact to define the encapsulation efficiency and release rate of nanoparticles formulated from 50:50 PLGA. A midrange molecular weight polymer had more desirable release properties at 100 mg/mL than at 50 mg/mL protein loading, indicating the need for optimization of nanoparticle formulation for preparations with different particle loadings. Mixtures of proteins derived from cell lysates were reliably encapsulated into nanoparticles, which released the spectrum of proteins contained in lysates. Antigenic proteins were co-encapsulated with cell lysate and released from nanoparticles; these Ags retained their antigenicity and functioned better than soluble Ags when tested in in vitro assays of T cell cytokine formation and in vivo tumor vaccination challenge.

Glioblastoma multiforme (GBM) is a fatal brain tumor characterized by infiltration beyond the margins of the main tumor mass and local recurrence after surgery. The blood-brain barrier (BBB) poses the most significant hurdle to brain tumor treatment. Convection-enhanced delivery (CED) allows for local administration of agents, overcoming the restrictions of the BBB. Recently, polymer nanoparticles have been demonstrated to penetrate readily through the healthy brain when delivered by CED, and size has been shown to be a critical factor for nanoparticle penetration. Because these brain-penetrating nanoparticles (BPNPs) have high potential for treatment of intracranial tumors since they offer the potential for cell targeting and controlled drug release after administration, here we investigated the intratumoral CED infusions of PLGA BPNPs in animals bearing either U87 or RG2 intracranial tumors. We demonstrate that the overall volume of distribution of these BPNPs was similar to that observed in healthy brains; however, the presence of tumors resulted in asymmetric and heterogeneous distribution patterns, with substantial leakage into the peritumoral tissue. Together, our results suggest that CED of BPNPs should be optimized by accounting for tumor geometry, in terms of location, size and presence of necrotic regions, to determine the ideal infusion site and parameters for individual tumors.

Intracranial implants elicit neurodegeneration via the foreign body response (FBR) that includes BBB leakage, macrophage/microglia accumulation, and reactive astrogliosis, in addition to neuronal degradation that limit their useful lifespan. Previously, monocyte chemoattractant protein 1 (MCP-1, also CCL2), which plays an important role in monocyte recruitment and propagation of inflammation, was shown to be critical for various aspects of the FBR in a tissue-specific manner. However, participation of MCP-1 in the brain FBR has not been evaluated. Here we examined the FBR to intracortical silicon implants in MCP-1 KO mice at 1, 2, and 8 weeks after implantation. MCP-1 KO mice had a diminished FBR compared to WT mice, characterized by reductions in BBB leakage, macrophage/microglia accumulation, and astrogliosis, and an increased neuronal density. Moreover, pharmacological inhibition of MCP-1 in implant-bearing WT mice maintained the increased neuronal density. To elucidate the relative contribution of microglia and macrophages, bone marrow chimeras were generated between MCP-1 KO and WT mice. Increased neuronal density was observed only in MCP-1 knockout mice transplanted with MCP-1 knockout marrow, which indicates that resident cells in the brain are major contributors. We hypothesized that these improvements are the result of a phenotypic switch of the macrophages/microglia polarization state, which we confirmed using PCR for common activation markers. Our observations suggest that MCP-1 influences neuronal loss, which is integral to the progression of neurological disorders like Alzheimer’s and Parkinson disease, via BBB leakage and macrophage polarization.