Santosh Aryal

Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform

Efforts to extend nanoparticle residence time in vivo have inspired many strategies in particle surface modifications to bypass macrophage uptake and systemic clearance. Here we report a top-down biomimetic approach in particle functionalization by coating biodegradable polymeric nanoparticles with natural erythrocyte membranes, including both membrane lipids and associated membrane proteins for long-circulating cargo delivery. The structure, size and surface zeta potential, and protein contents of the erythrocyte membrane-coated nanoparticles were verified using transmission electron microscopy, dynamic light scattering, and gel electrophoresis, respectively. Mice injections with fluorophore-loaded nanoparticles revealed superior circulation half-life by the erythrocyte-mimicking nanoparticles as compared to control particles coated with the state-of-the-art synthetic stealth materials. Biodistribution study revealed significant particle retention in the blood 72 h following the particle injection. The translocation of natural cellular membranes, their associated proteins, and the corresponding functionalities to the surface of synthetic particles represents a unique approach in nanoparticle functionalization.

Polymeric Nanoparticles with Precise Ratiometric Control over Drug Loading for Combination Therapy

We report a novel approach for nanoparticle-based combination chemotherapy by concurrently incorporating two different types of drugs into a single polymeric nanoparticle with ratiometric control over the loading of the two drugs. By adapting metal alkoxide chemistry, we synthesize highly hydrophobic drug-poly-L-lactide (drug-PLA) conjugates, of which the polymer has the same chain length while the drug may differ. These drug-polymer conjugates are then encapsulated into lipid-coated polymeric nanoparticles through a single-step nanoprecipitation method. Using doxorubicin (DOX) and camptothecin (CPT) as two model chemotherapy drugs, various ratios of DOX-PLA and CPT-PLA conjugates are loaded into the nanoparticles with over 90% loading efficiency. The resulting nanoparticles are uniform in size, size distribution and surface charge. The loading yield of DOX and CPT in the particles can be precisely controlled by simply adjusting the DOX-PLA:CPT-PLA molar ratio. Cellular cytotoxicity results show that the dual-drug loaded nanoparticles are superior to the corresponding cocktail mixtures of single-drug loaded nanoparticles. This dual-drug delivery approach offers a solution to the long-standing challenge in ratiometric control over the loading of different types of drugs onto the same drug delivery vehicle. We expect that this approach can be exploited for many types of chemotherapeutic agents containing hydroxyl groups and thus enable codelivery of various drug combinations for combinatorial treatments of diseases.

Stimuli-responsive liposome fusion mediated by gold nanoparticles.

We report a new approach to controlling the fusion activity of liposomes by adsorbing carboxyl-modified gold nanoparticles to the outer surface of phospholipid liposomes. The bound gold nanoparticles can effectively prevent liposomes from fusing with one another at neutral pH value, while at acidic environments (e.g., pH < 5), the gold particle stabilizers will detach from the liposomes, with liposome fusion activity resuming. The binding of carboxyl-modified gold nanoparticles to cationic phospholipid liposomes at neutral pH and detaching at acidic pH values are evaluated and confirmed by dynamic light scattering, electron microscopy, fluorescence and UV-vis absorption experiments. The relative fusion efficiency of gold-nanoparticle-stabilized cationic liposomes with anionic liposomes is approximately 25% at pH = 7 in contrast to approximately 80% at pH = 4. Since liposomes have been extensively used as drug nanocarriers and the infectious lesions on human skin are typically acidic with a pH < 5, these acid-responsive liposomes with tunable fusion ability hold great promise for dermal drug delivery to treat a variety of skin diseases such as acne vulgaris and staph infections.

Erythrocyte membrane-cloaked polymeric nanoparticles for controlled drug loading and release

AIM Polymeric nanoparticles (NPs) cloaked by red blood cell membrane (RBCm) confer the combined advantage of both long circulation lifetime and controlled drug release. The authors carried out studies to gain a better understanding of the drug loading, drug-release kinetics and cell-based efficacy of RBCm-cloaked NPs. MATERIALS & METHODS Two strategies for loading doxorubicin into the RBCm-cloaked NPs were compared: physical encapsulation and chemical conjugation. In vitro efficacy was examined using the acute myeloid leukemia cell line, Kasumi-1. RESULTS It was found that the chemical conjugation strategy resulted in a more sustained drug release profile, and that the RBCm cloak provided a barrier, retarding the outward diffusion of encapsulated drug molecules. It was also demonstrated that RBCm-cloaked NPs exhibit higher toxicity in comparison with free doxorubicin. CONCLUSION These results indicate that the RBCm-cloaked NPs hold great promise to become a valuable drug-delivery platform for the treatment of various diseases such as blood cancers.

Soft Discoidal Polymeric Nanoconstructs Resist Macrophage Uptake and Enhance Vascular Targeting in Tumors

Most nanoparticles for biomedical applications originate from the self-assembling of individual constituents through molecular interactions and possess limited geometry control and stability. Here, 1000 × 400 nm discoidal polymeric nanoconstructs (DPNs) are demonstrated by mixing hydrophobic and hydrophilic polymers with lipid chains and curing the resulting paste directly within silicon templates. By changing the paste composition, soft- and rigid-DPNs (s- and r-DPNs) are synthesized exhibiting the same geometry, a moderately negative surface electrostatic charge (-14 mV), and different mechanical stiffness (∼1.3 and 15 kPa, respectively). Upon injection in mice bearing nonorthotopic brain or skin cancers, s-DPNs exhibit ∼24 h circulation half-life and accumulate up to ∼20% of the injected dose per gram tumor, detecting malignant masses as small as ∼0.1% the animal weight via PET imaging. This unprecedented behavior is ascribed to the unique combination of geometry, surface properties, and mechanical stiffness which minimizes s-DPN sequestration by the mononuclear phagocyte system. Our results could boost the interest in using less conventional delivery systems for cancer theranosis.

Engineered magnetic hybrid nanoparticles with enhanced relaxivity for tumor imaging

Clinically used contrast agents for magnetic resonance imaging (MRI) suffer by the lack of specificity; short circulation time; and insufficient relaxivity. Here, a one-step combinatorial approach is described for the synthesis of magnetic lipid-polymer (hybrid) nanoparticles (MHNPs) encapsulating 5 nm ultra-small super-paramagnetic iron oxide particles (USPIOs) and decorated with Gd(3+) ions. The MHNPs comprise a hydrophobic poly(lactic acid-co-glycolic acid) (PLGA) core, containing up to ~5% USPIOs (w/w), stabilized by lipid and polyethylene glycol (PEG). Gd(3+) ions are directly chelated to the external lipid monolayer. Three different nanoparticle configurations are presented including Gd(3+) chelates only (Gd-MHNPs); USPIOs only (Fe-MHNPs); and the combination thereof (MHNPs). All three MHNPs exhibit a hydrodynamic diameter of about 150 nm. The Gd-MHNPs present a longitudinal relaxivity (r1 = 12.95 ± 0.53 (mM s)(-1)) about four times larger than conventional Gd-based contrast agents (r1 = 3.4 (mM s)(-1)); MHNPs have a transversal relaxivity of r2 = 164.07 ± 7.0 (mM s)(-1), which is three to four times larger than most conventional systems (r2 ~ 50 (mM s)(-1)). In melanoma bearing mice, elemental analysis for Gd shows about 3% of the injected MHNPs accumulating in the tumor and 2% still circulating in the blood, at 24 h post-injection. In a clinical 3T MRI scanner, MHNPs provide significant contrast confirming the observed tumor deposition. This approach can also accommodate the co-loading of hydrophobic therapeutic compounds in the MHNP core, paving the way for theranostic systems.

Positron emitting magnetic nanoconstructs for PET/MR imaging.

Hybrid PET/MRI scanners have the potential to provide fundamental molecular, cellular, and anatomic information essential for optimizing therapeutic and surgical interventions. However, their full utilization is currently limited by the lack of truly multi-modal contrast agents capable of exploiting the strengths of each modality. Here, we report on the development of long-circulating positron-emitting magnetic nanoconstructs (PEM) designed to image solid tumors for combined PET/MRI. PEMs are synthesized by a modified nano-precipitation method mixing poly(lactic-co-glycolic acid) (PLGA), lipids, and polyethylene glycol (PEG) chains with 5 nm iron oxide nanoparticles (USPIOs). PEM lipids are coupled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and subsequently chelated to (64)Cu. PEMs show a diameter of 140 ± 7 nm and a transversal relaxivity r2 of 265.0 ± 10.0 (mM × s)(-1), with a r2/r1 ratio of 123. Using a murine xenograft model bearing human breast cancer cell line (MDA-MB-231), intravenously administered PEMs progressively accumulate in tumors reaching a maximum of 3.5 ± 0.25% ID/g tumor at 20 h post-injection. Correlation of PET and MRI signals revealed non-uniform intratumoral distribution of PEMs with focal areas of accumulation at the tumor periphery. These long-circulating PEMs with high transversal relaxivity and tumor accumulation may allow for detailed interrogation over multiple scales in a clinically relevant setting.

Large-scale synthesis of lipid-polymer hybrid nanoparticles using a multi-inlet vortex reactor.

Lipid-polymer hybrid nanoparticles combine the advantages of both polymeric and liposomal drug carriers and have shown great promise as a controlled drug delivery platform. Herein, we demonstrate that it is possible to adapt a multi-inlet vortex reactor (MIVR) for use in the large-scale synthesis of these hybrid nanoparticles. Several parameters, including formulation, polymer concentration, and flow rate, are systematically varied, and the effects of each on nanoparticle properties are studied. Particles fabricated from this process display characteristics that are on par with those made on the lab-scale such as small size, low polydispersity, and excellent stability in both PBS and serum. Using this approach, production rates of greater than 10 g/h can readily be achieved, demonstrating that use of the MIVR is a viable method of producing hybrid nanoparticles in clinically relevant quantities.

Synthesis of multifunctional magnetic nanoflakes for magnetic resonance imaging, hyperthermia, and targeting.

Iron oxide nanoparticles (IOs) are intrinsically theranostic agents that could be used for magnetic resonance imaging (MRI) and local hyperthermia or tissue thermal ablation. Yet, effective hyperthermia and high MR contrast have not been demonstrated within the same nanoparticle configuration. Here, magnetic nanoconstructs are obtained by confining multiple, ∼ 20 nm nanocubes (NCs) within a deoxy-chitosan core. The resulting nanoconstructs—magnetic nanoflakes (MNFs)—exhibit a hydrodynamic diameter of 156 ± 3.6 nm, with a polydispersity index of ∼0.2, and are stable in PBS up to 7 days. Upon exposure to an alternating magnetic field of 512 kHz and 10 kA m–1, MNFs provide a specific absorption rate (SAR) of ∼75 W gFe–1, which is 4–15 times larger than that measured for conventional IOs. Moreover, the same nanoconstructs provide a remarkably high transverse relaxivity of ∼500 (mM s)−1, at 1.41T. MNFs represent a first step toward the realization of nanoconstructs with superior relaxometric and ablation properties for more effective theranostics.

Engineering discoidal polymeric nanoconstructs with enhanced magneto-optical properties for tumor imaging.

The in vivo performance of nanoparticles is affected by their size, shape and surface properties. Fabrication methods based on emulsification and nano-precipitation cannot control these features precisely and independently over multiple scales. Herein, discoidal polymeric nanoconstructs (DPNs) with a diameter of 1000 nm and a height of 500 nm are demonstrated via a modified hydrogel-template strategy. The DPNs are obtained by mixing in one synthesis step the constituent polymers - poly(lactic acid-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) dimethacrylate - and the payload with magneto-optical properties - 5 nm ultra-small super-paramagnetic iron oxide nanoparticles (SPIOs) and Rhodamine B dye (RhB). The DPN geometrical features are characterized by multiple microscopy techniques. The release of the Rhodamine B dye is pH dependent and increases under acidic conditions by the enhanced hydrolysis of the polymeric matrix. Each DPN is loaded with ~100 fg of iron and can be efficiently dragged by static and external magnetic fields. Moreover, the USPIO confinement within the DPN porous structure is responsible for a significant enhancement in MRI relaxivity (r2 ~ 500 (mMs)(-1)), up to ~5 times larger than commercially available systems. These nanoconstructs suggest a general strategy to engineer theranostic systems for anti-angiogenic treatment and vascular imaging.