Docetaxel-Loaded PLGA Nanoparticles Improve Efficacy in Taxane-Resistant Triple-Negative Breast Cancer.

Novel treatment strategies, including nanomedicine, are needed for improving management of triple-negative breast cancer. Patients with triple-negative breast cancer, when considered as a group, have a worse outcome after chemotherapy than patients with breast cancers of other subtypes, a finding that reflects the intrinsically adverse prognosis associated with the disease. The aim of this study was to improve the efficacy of docetaxel by incorporation into a novel nanoparticle platform for the treatment of taxane-resistant triple-negative breast cancer. Rod-shaped nanoparticles encapsulating docetaxel were fabricated using an imprint lithography based technique referred to as Particle Replication in Nonwetting Templates (PRINT). These rod-shaped PLGA-docetaxel nanoparticles were tested in the C3(1)-T-antigen (C3Tag) genetically engineered mouse model (GEMM) of breast cancer that represents the basal-like subtype of triple-negative breast cancer and is resistant to therapeutics from the taxane family. This GEMM recapitulates the genetics of the human disease and is reflective of patient outcome and, therefore, better represents the clinical impact of new therapeutics. Pharmacokinetic analysis showed that delivery of these PLGA-docetaxel nanoparticles increased docetaxel circulation time and provided similar docetaxel exposure to tumor compared to the clinical formulation of docetaxel, Taxotere. These PLGA-docetaxel nanoparticles improved tumor growth inhibition and significantly increased median survival time. This study demonstrates the potential of nanotechnology to improve the therapeutic index of chemotherapies and rescue therapeutic efficacy to treat nonresponsive cancers.

Particle replication in nonwetting templates nanoparticles with tumor selective alkyl silyl ether docetaxel prodrug reduces toxicity.

Delivery systems designed to have triggered release after passively targeting the tumor may improve small molecule chemotherapeutic delivery. Particle replication in nonwetting templates was used to prepare nanoparticles to passively target solid tumors in an A549 subcutaneous xenograft model. An acid labile prodrug was delivered to minimize systemic free docetaxel concentrations and improve tolerability without compromising efficacy.

RNA replicon delivery via lipid-complexed PRINT protein particles.

Herein we report the development of a nonviral lipid-complexed PRINT (particle replication in nonwetting templates) protein particle system (LPP particle) for RNA replicon delivery with a view toward RNA replicon-based vaccination. Cylindrical bovine serum albumin (BSA) particles (diameter (d) 1 µm, height (h) 1 µm) loaded with RNA replicon and stabilized with a fully reversible disulfide cross-linker were fabricated using PRINT technology. Highly efficient delivery of the particles to Vero cells was achieved by complexing particles with a mixture of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids. Our data suggest that (1) this lipid-complexed protein particle is a promising system for delivery of RNA replicon-based vaccines and (2) it is necessary to use a degradable cross-linker for successful delivery of RNA replicon via protein-based particles.

Plasma, tumor and tissue pharmacokinetics of Docetaxel delivered via nanoparticles of different sizes and shapes in mice bearing SKOV-3 human ovarian carcinoma xenograft.

The particle fabrication technique PRINT® was used to fabricate monodisperse size and shape specific poly(lactide-co-glycolide) particles loaded with the chemotherapeutic Docetaxel. The pharmacokinetics of two cylindrical shaped particles with diameter=80nm; height=320nm (PRINT-Doc-80×320) and d=200nm; h=200nm (PRINT-Doc-200×200) were compared to Docetaxel in mice bearing human ovarian carcinoma SKOV-3 flank xenografts. The Docetaxel plasma exposure was ~20-fold higher for both particles compared to docetaxel. Additionally, the volume of distribution (Vd) of Docetaxel in PRINT formulations was ~18-fold (PRINT-Doc-80×320) and ~33-fold (PRINT-Doc-200×200) lower than Docetaxel. The prolonged duration of Docetaxel in plasma when dosed with PRINT formulations subsequently led to increased tumor exposure of Docetaxel from 0 to 168h (~53% higher for PRINT-Doc-80×320 and ~76% higher for PRINT-Doc-200×200 particles). PRINT-Doc-80×320 had lower exposures in the liver, spleen and lung compared with PRINT-Doc-200×200. Thus, the use of particles with smaller feature size may be preferred to decrease clearance by organs of the mononuclear phagocyte system. FROM THE CLINICAL EDITOR: In this study, the plasma, tumor, and tissue pharmacokinetics of different Docetaxel nanoparticles of precise shape and size were characterized in mice with human ovarian carcinoma xenograft. It is concluded that the use of particles with smaller feature size may be preferred to decrease clearance by organs of the mononuclear phagocyte system.

Delivery of multiple siRNAs using lipid-coated PLGA nanoparticles for treatment of prostate cancer.

Nanotechnology can provide a critical advantage in developing strategies for cancer management and treatment by helping to improve the safety and efficacy of novel therapeutic delivery vehicles. This paper reports the fabrication of poly(lactic acid-co-glycolic acid)/siRNA nanoparticles coated with lipids for use as prostate cancer therapeutics made via a unique soft lithography particle molding process called Particle Replication In Nonwetting Templates (PRINT). The PRINT process enables high encapsulation efficiency of siRNA into neutral and monodisperse PLGA particles (32-46% encapsulation efficiency). Lipid-coated PLGA/siRNA PRINT particles were used to deliver therapeutic siRNA in vitro to knockdown genes relevant to prostate cancer.

Incorporation and controlled release of silyl ether prodrugs from PRINT nanoparticles.

Asymmetric bifunctional silyl ether (ABS) prodrugs of chemotherapeutics were synthesized and incorporated within 200 nm × 200 nm particles. ABS prodrugs of gemcitabine were selected as model compounds because of the difficulty to encapsulate a water-soluble drug within a hydrogel. The resulting drug delivery systems were degraded under acidic conditions and were found to release only the parent or active drug. Furthermore, changing the steric bulk of the alkyl substituents on the silicon atom could regulate the rate of drug release and, therefore, the intracellular toxicity of the gemcitabine-loaded particles. This yielded a family of novel nanoparticles that could be tuned to release drug over the course of hours, days, or months.

Rendering protein-based particles transiently insoluble for therapeutic applications.

Herein, we report the fabrication of protein (bovine serum albumin, BSA) particles which were rendered transiently insoluble using a novel, reductively labile disulfide-based cross-linker. After being cross-linked, the protein particles retain their integrity in aqueous solution and dissolve preferentially under a reducing environment. Our data demonstrates that cleavage of the cross-linker leaves no chemical residue on the reactive amino group. Delivery of a self-replicating RNA was achieved via the transiently insoluble PRINT protein particles. These protein particles can provide new opportunities for drug and gene delivery.

Reductively responsive siRNA-conjugated hydrogel nanoparticles for gene silencing.

A critical need still remains for effective delivery of RNA interference (RNAi) therapeutics to target tissues and cells. Self-assembled lipid- and polymer-based systems have been most extensively explored for transfection with small interfering RNA (siRNA) in liver and cancer therapies. Safety and compatibility of materials implemented in delivery systems must be ensured to maximize therapeutic indices. Hydrogel nanoparticles of defined dimensions and compositions, prepared via a particle molding process that is a unique off-shoot of soft lithography known as particle replication in nonwetting templates (PRINT), were explored in these studies as delivery vectors. Initially, siRNA was encapsulated in particles through electrostatic association and physical entrapment. Dose-dependent gene silencing was elicited by PEGylated hydrogels at low siRNA doses without cytotoxicity. To prevent disassociation of cargo from particles after systemic administration or during postfabrication processing for surface functionalization, a polymerizable siRNA pro-drug conjugate with a degradable, disulfide linkage was prepared. Triggered release of siRNA from the pro-drug hydrogels was observed under a reducing environment while cargo retention and integrity were maintained under physiological conditions. Gene silencing efficiency and cytocompatibility were optimized by screening the amine content of the particles. When appropriate control siRNA cargos were loaded into hydrogels, gene knockdown was only encountered for hydrogels containing releasable, target-specific siRNAs, accompanied by minimal cell death. Further investigation into shape, size, and surface decoration of siRNA-conjugated hydrogels should enable efficacious targeted in vivo RNAi therapies.

PRINT: a novel platform toward shape and size specific nanoparticle theranostics.

Nanotheranostics represents the next generation of medicine, fusing nanotechnology, therapeutics, and diagnostics. By integrating therapeutic and imaging agents into one nanoparticle, this new treatment strategy has the potential not only to detect and diagnose disease but also to treat and monitor the therapeutic response. This capability could have a profound impact in both the research setting as well as in a clinical setting. In the research setting, such a capability will allow research scientists to rapidly assess the performance of new therapeutics in an effort to iterate their designs for increased therapeutic index and efficacy. In the clinical setting, theranostics offers the ability to determine whether patients enrolling in clinical trials are responding, or are expected to respond, to a given therapy based on the hypothesis associated with the biological mechanisms being tested. If not, patients can be more quickly removed from the clinical trial and shifted to other therapeutic options. To be effective, these theranostic agents must be highly site specific. Optimally, they will carry relevant cargo, demonstrate controlled release of that cargo, and include imaging probes with a high signal-to-noise ratio. There are many biological barriers in the human body that challenge the efficacy of nanoparticle delivery vehicles. These barriers include, but are not limited to, the walls of blood vessels, the physical entrapment of particles in organs, and the removal of particles by phagocytic cells. The rapid clearance of circulating particles during systemic delivery is a major challenge; current research seeks to define key design parameters that govern the performance of nanocarriers, such as size, surface chemistry, elasticity, and shape. The effect of particle size and surface chemistry on in vivo biodistribution of nanocarriers has been extensively studied, and general guidelines have been established. Recently it has been documented that shape and elasticity can have a profound effect on the behavior of delivery vehicles. Thus, having the ability to independently control shape, size, matrix, surface chemistry, and modulus is crucial for designing successful delivery agents. In this Account, we describe the use of particle replication in nonwetting templates (PRINT) to fabricate shape- and size-specific microparticles and nanoparticles. A particular strength of the PRINT method is that it affords precise control over shape, size, surface chemistry, and modulus. We have demonstrated the loading of PRINT particles with chemotherapeutics, magnetic resonance contrast agents, and fluorophores. The surface properties of the PRINT particles can be easily modified with "stealth" poly(ethylene glycol) chains to increase blood circulation time, with targeting moieties for targeted delivery or with radiolabels for nuclear imaging. These particles have tremendous potential for applications in nanomedicine and diagnostics.

Effect of aspect ratio and deformability on nanoparticle extravasation through nanopores.

We describe the fabrication of filamentous hydrogel nanoparticles using a unique soft lithography based particle molding process referred to as PRINT (particle replication in nonwetting templates). The nanoparticles possess a constant width of 80 nm, and we varied their lengths ranging from 180 to 5000 nm. In addition to varying the aspect ratio of the particles, the deformability of the particles was tuned by varying the cross-link density within the particle matrix. Size characteristics such as hydrodynamic diameter and persistence length of the particles were analyzed using dynamic light scattering and electron microscopy techniques, respectively, while particle deformability was assessed by atomic force microscopy. Additionally, the ability of the particles to pass through membranes containing 0.2 µm pores was assessed by means of a simple filtration technique, and particle recovery was determined using fluorescence spectroscopy. The results show that particle recovery is mostly independent of aspect ratio at all cross-linker concentrations utilized, with the exception of 96 wt % PEG diacrylate 80 × 5000 nm particles, which showed the lowest percent recovery.

Low modulus biomimetic microgel particles with high loading of hemoglobin.

We synthesized extremely deformable red blood cell-like microgel particles and loaded them with bovine hemoglobin (Hb) to potentiate oxygen transport. With similar shape and size as red blood cells (RBCs), the particles were fabricated using the PRINT (particle replication in nonwetting templates) technique. Low cross-linking of the hydrogel resulted in very low mesh density for these particles, allowing passive diffusion of hemoglobin throughout the particles. Hb was secured in the particles through covalent conjugation of the lysine groups of Hb to carboxyl groups in the particles via EDC/NHS coupling. Confocal microscopy of particles bound to fluorescent dye-labeled Hb confirmed the uniform distribution of Hb throughout the particle interior, as opposed to the surface conjugation only. High loading ratios, up to 5 times the amount of Hb to polymer by weight, were obtained without a significant effect on particle stability and shape, though particle diameter decreased slightly with Hb conjugation. Analysis of the protein by circular dichroism (CD) spectroscopy showed that the secondary structure of Hb was unperturbed by conjugation to the particles. Methemoglobin in the particles could be maintained at a low level and the loaded Hb could still bind oxygen, as studied by UV-vis spectroscopy. Hb-loaded particles with moderate loading ratios demonstrated excellent deformability in microfluidic devices, easily deforming to pass through restricted pores half as wide as the diameter of the particles. The suspension of concentrated particles with a Hb concentration of 5.2 g/dL showed comparable viscosity to that of mouse blood, and the particles remained intact even after being sheared at a constant high rate (1000 1/s) for 10 min. Armed with the ability to control size, shape, deformability, and loading of Hb into RBC mimics, we will discuss the implications for artificial blood.

Potent engineered PLGA nanoparticles by virtue of exceptionally high chemotherapeutic loadings.

Herein we report the fabrication of engineered poly(lactic acid-co-glycolic acid) nanoparticles via the PRINT (particle replication in nonwetting templates) process with high and efficient loadings of docetaxel, up to 40% (w/w) with encapsulation efficiencies >90%. The PRINT process enables independent control of particle properties leading to a higher degree of tailorability than traditional methods. Particles with 40% loading display better in vitro efficacy than particles with lower loadings and the clinical formulation of docetaxel, Taxotere.

More effective nanomedicines through particle design.

Nanomedicine is an emerging field that applies concepts in nanotechnology to develop novel diagnostics and therapies. Physical and chemical properties of particles, including size, shape, modulus, surface charge and surface chemistry, play an important role in determining particle-cell interactions, cellular trafficking mechanisms, biodistribution, and pharmacokinetics. This discussion focuses on both nanoparticles and microparticles since microparticles can also provide many insights for the development of drug carriers and possess advantages over nanoparticles in certain applications. This review covers recent major advancement in the nanomedicine field and also highlights studies using the PRINT technology.

Novel platforms for vascular carriers with controlled geometry.

The first-generation platforms for vascular drug delivery adopted spherical morphologies. These carriers relied primarily on the size dependence of the enhanced permeability and retention effect to passively target vasculature, resulting in inefficient delivery due to significant variation in endothelial permeability. Enhanced delivery typically requires active targeting via receptor-mediated endocytosis by surface conjugation of targeting ligands. However, vascular carriers (VCs) still face numerous challenges en route to reaching their targets before delivery. The control of carrier shape offers opportunities to overcome in vivo barriers and enhance vascular drug delivery. Geometric features influence the ability of carrier particles to navigate physiological flow patterns, evade biological clearance mechanisms, sustain circulation, adhere to the vascular surface, and finally transport across or internalize into the endothelium. Although previous formulation strategies limited the fabrication of nonspherical carriers, numerous recent advances in both top-down and bottom-up fabrication techniques have enabled shape modulation as a key design element. As part of a series on vascular drug delivery, this review focuses on recent developments in novel vascular platforms with controlled geometry that enhance or modulate delivery functions. Starting with an overview of controlled geometry platforms, we review their shape-dependent functional characteristics for each stage of their vascular journey in vivo. We sequentially explore carrier geometries that evade reticuloendothelial system uptake, display enhanced circulation persistence and margination dynamics in flow, encourage adhesion to the vascular surface or extravasation through endothelium, and impact extravascular transport and cell internalization. The eventual biodistribution of VCs results from the culmination of their successive navigation of all these barriers and is profoundly influenced by their morphology. To enhance delivery efficacy, carrier designs synergistically combining controlled geometry with standard drug delivery strategies such as targeting moieties, surface decorations, and bulk material properties are discussed. Finally, we speculate on possibilities for innovation, harnessing shape as a design parameter for the next generation of vascular drug delivery platforms.

The effect of particle design on cellular internalization pathways.

The interaction of particles with cells is known to be strongly influenced by particle size, but little is known about the interdependent role that size, shape, and surface chemistry have on cellular internalization and intracellular trafficking. We report on the internalization of specially designed, monodisperse hydrogel particles into HeLa cells as a function of size, shape, and surface charge. We employ a top-down particle fabrication technique called PRINT that is able to generate uniform populations of organic micro- and nanoparticles with complete control of size, shape, and surface chemistry. Evidence of particle internalization was obtained by using conventional biological techniques and transmission electron microscopy. These findings suggest that HeLa cells readily internalize nonspherical particles with dimensions as large as 3 mum by using several different mechanisms of endocytosis. Moreover, it was found that rod-like particles enjoy an appreciable advantage when it comes to internalization rates, reminiscent of the advantage that many rod-like bacteria have for internalization in nonphagocytic cells.

The complex role of multivalency in nanoparticles targeting the transferrin receptor for cancer therapies.

Transferrin receptor (TfR, CD71) has long been a therapeutic target due to its overexpression in many malignant tissues. In this study, PRINT() nanoparticles were conjugated with TfR ligands for targeted drug delivery. Cylindrical poly(ethylene glycol)-based PRINT nanoparticles (diameter (d) = 200 nm, height (h) = 200 nm) labeled with transferrin receptor antibody (NP-OKT9) or human transferrin (NP-hTf) showed highly specific TfR-mediated uptake by all human tumor cell lines tested, relative to negative controls (IgG1 for OKT9 or bovine transferrin (bTf) for hTf). The targeting efficiency was dependent on particle concentration, ligand density, dosing time, and cell surface receptor expression level. Interestingly, NP-OKT9 or NP-hTf showed little cytotoxicity on all solid tumor cell lines tested but were very toxic to Ramos B-cell lymphoma, whereas free OKT9 or hTf was not toxic. There was a strong correlation between TfR ligand density on the particle surface and cell viability and particle uptake. NP-OKT9 and NP-hTf were internalized into acidic intracellular compartments but were not localized in EEA1-enriched early endosomes or lysosomes. Elevated caspase 3/7 activity indicates activation of apoptosis pathways upon particle treatment. Supplementation of iron suppressed the toxicity of NP-OKT9 but not NP-hTf, suggesting different mechanisms by which NP-hTf and NP-OKT9 exerts cytotoxicity on Ramos cells. On the basis of such an observation, the complex role of multivalency in nanoparticles is discussed. In addition, our data clearly reveal that one must be careful in making claims of "lack of toxicity" when a targeting molecule is used on nanoparticles and also raise concerns for unanticipated off-target effects when one is designing targeted chemotherapy nanodelivery agents.

Tunable bifunctional silyl ether cross-linkers for the design of acid-sensitive biomaterials.

Responsive polymeric biomaterials can be triggered to degrade using localized environments found in vivo. A limited number of biomaterials provide precise control over the rate of degradation and the release rate of entrapped cargo and yield a material that is intrinsically nontoxic. In this work, we designed nontoxic acid-sensitive biomaterials based on silyl ether chemistry. A host of silyl ether cross-linkers were synthesized and molded into relevant medical devices, including Trojan horse particles, sutures, and stents. The resulting devices were engineered to degrade under acidic conditions known to exist in tumor tissue, inflammatory tissue, and diseased cells. The implementation of silyl ether chemistry gave precise control over the rate of degradation and afforded devices that could degrade over the course of hours, days, weeks, or months, depending upon the steric bulk around the silicon atom. These novel materials could be useful for numerous biomedical applications, including drug delivery, tissue repair, and general surgery.

The pursuit of a scalable nanofabrication platform for use in material and life science applications.

In this Account, we describe the use of perfluoropolyether (PFPE)-based materials that are able to accurately mold and replicate micro- and nanosized features using traditional techniques such as embossing as well as new techniques that we developed to exploit the exceptional surface characteristics of fluorinated substrates. Because of the unique partial wetting and nonwetting characteristics of PFPEs, we were able to go beyond the usual molding and imprint lithography approaches and have created a technique called PRINT (Particle [or Pattern] Replication In Nonwetting Templates). PRINT is a distinctive "top-down" fabrication technique capable of generating isolated particles, arrays of particles, and arrays of patterned features for a plethora of applications in both nanomedicine and materials science. A particular strength of the PRINT technology is the high-resolution molding of well-defined particles with precise control over size, shape, deformability, and surface chemistry. The level of replication obtained showcases some of the unique characteristics of PFPE molding materials. In particular, these materials arise from very low surface energy precursors with positive spreading coefficients, can be photocured at ambient temperature, and are minimally adhesive, nonswelling, and conformable. These distinctive features enable the molding of materials with unique attributes and nanometer resolution that have unprecedented scientific and technological value. For example, in nanomedicine, the use of PFPE materials with the PRINT technique allows us to design particles in which we can tailor key therapeutic parameters such as bioavailability, biodistribution, target-specific cell penetration, and controlled cargo release. Similarly, in materials science, we can fabricate optical films and lens arrays, replicate complex, naturally occurring objects such as adenovirus particles, and create 2D patterned arrays of inorganic oxides.

Local iontophoretic administration of cytotoxic therapies to solid tumors.

Parenteral and oral routes have been the traditional methods of administering cytotoxic agents to cancer patients. Unfortunately, the maximum potential effect of these cytotoxic agents has been limited because of systemic toxicity and poor tumor perfusion. In an attempt to improve the efficacy of cytotoxic agents while mitigating their side effects, we have developed modalities for the localized iontophoretic delivery of cytotoxic agents. These iontophoretic devices were designed to be implanted proximal to the tumor with external control of power and drug flow. Three distinct orthotopic mouse models of cancer and a canine model were evaluated for device efficacy and toxicity. Orthotopic patient-derived pancreatic cancer xenografts treated biweekly with gemcitabine via the device for 7 weeks experienced a mean log2 fold change in tumor volume of -0.8 compared to a mean log2 fold change in tumor volume of 1.1 for intravenous (IV) gemcitabine, 3.0 for IV saline, and 2.6 for device saline groups. The weekly coadministration of systemic cisplatin therapy and transdermal device cisplatin therapy significantly increased tumor growth inhibition and doubled the survival in two aggressive orthotopic models of breast cancer. The addition of radiotherapy to this treatment further extended survival. Device delivery of gemcitabine in dogs resulted in more than 7-fold difference in local drug concentrations and 25-fold lower systemic drug levels than the IV treatment. Overall, these devices have potential paradigm shifting implications for the treatment of pancreatic, breast, and other solid tumors.

Metronomic docetaxel in PRINT nanoparticles and EZH2 silencing have synergistic antitumor effect in ovarian cancer.

The purpose of this study was to investigate the antitumor effects of a combination of metronomic doses of a novel delivery vehicle, PLGA-PRINT nanoparticles containing docetaxel, and antiangiogenic mEZH2 siRNA incorporated into chitosan nanoparticles. In vivo dose-finding studies and therapeutic experiments were conducted in well-established orthotopic mouse models of epithelial ovarian cancer. Antitumor effects were determined on the basis of reduction in mean tumor weight and number of metastatic tumor nodules in the animals. The tumor tissues from these in vivo studies were stained to evaluate the proliferation index (Ki67), apoptosis index (cleaved caspase 3), and microvessel density (CD31). The lowest dose of metronomic regimen (0.5 mg/kg) resulted in significant reduction in tumor growth. The combination of PLGA-PRINT-docetaxel and CH-mEZH2 siRNA showed significant antitumor effects in the HeyA8 and SKOV3ip1 tumor models (P < 0.05). Individual as well as combination therapies showed significant antiangiogenic, antiproliferative, and proapoptotic effects, and combination therapy had additive effects. Metronomic delivery of PLGA-PRINT-docetaxel combined with CH-mEZH2 siRNA has significant antitumor activity in preclinical models of ovarian cancer.