A novel formulation enabled transformation of 3-HIV drugs tenofovir-lamivudine-dolutegravir from short-acting to long-acting all-in-one injectable.

OBJECTIVE: To develop an injectable dosage form of the daily oral HIV drugs, tenofovir (T), lamivudine (L), and dolutegravir (D), creating a single, complete, all-in-one TLD 3-drug-combination that demonstrates long-acting pharmacokinetics. DESIGN: Using drug-combination-nanoparticle (DcNP) technology to stabilize multiple HIV drugs, the 3-HIV drugs TLD, with disparate physical-chemical properties, are stabilized and assembled with lipid-excipients to form TLD-in-DcNP . TLD-in-DcNP is verified to be stable and suitable for subcutaneous administration. To characterize the plasma time-courses and PBMC concentrations for all 3 drugs, single subcutaneous injections of TLD-in-DcNP were given to nonhuman primates (NHP, M. nemestrina ). RESULTS: Following single-dose TLD-in-DcNP , all drugs exhibited long-acting profiles in NHP plasma with levels that persisted for 4 weeks above predicted viral-effective concentrations for TLD in combination. Times-to-peak were within 24 hr in all NHP for all drugs. Compared to a free-soluble TLD, TLD-in-DcNP provided exposure enhancement and extended duration 7.0-, 2.1-, and 20-fold as AUC boost and 10-, 8.3-, and 5.9-fold as half-life extension. Additionally, DcNP may provide more drug exposure in cells than plasma with PBMC-to-plasma drug ratios exceeding one, suggesting cell-targeted drug-combination delivery. CONCLUSIONS: This study confirms that TLD with disparate properties can be made stable by DcNP to enable TLD concentrations of 4 weeks in NHP. Study results highlighted the potential of TLD-in-DcNP as a convenient all-in-one, complete HIV long-acting product for clinical development.

Patterns of leukocyte recovery predict infectious complications after CD19 CAR-T cell therapy in a real-world setting.

BACKGROUND: Adoptive immunotherapy using CD19-targeted Chimeric antigen receptor T cells (CAR-T) has revolutionized the treatment of relapsed/refractory diffuse large B-cell lymphoma (DLBCL). Data is limited on the propensity of infections and lymphohematopoietic reconstitution after Day 30 (D30) following CAR-T cell therapy. In this study, we evaluated the prevalence and nature of infectious complications in an expanded cohort of DLBCL patients treated with CD19 CAR-T therapy and its association with the dynamics of leukocyte subpopulation reconstitution post-CAR-T cell therapy. METHODS: We conducted a retrospective study including 19 patients who received axicabtagene ciloleucel and investigated associations between cytopenia and infectious complications after D30. RESULTS: Nineteen patients were included, consisting of 42% Hispanic, 32% Caucasian, 21% African-American, and 5% Asian subjects. Post-D30 of CAR-T infusion, 47% patients (n=9) developed an infection and 53% (n=10) remained infection-free. The most common infection type observed was viral (7 patients) followed by bacterial (5 patients) and fungal (3 patients). Of 25 total infectious events, 56% were grade 1 or 2 and 44% were grade 3 with 10 being viral in etiology. To determine the kinetics of lymphohematopoietic reconstitution and its association with infection risk, we evaluated the relationship between cytopenias and rates of infection after D30. Notably, compared to non-infection group, infection group had a higher median absolute lymphocyte count (ALC) (1,000/µL vs. 600/µL, P<0.05), a lower median absolute neutrophil count (ANC)/ALC ratio (1.6 vs. 3.1, P<0.05) and a lower median AMC/ALC at D30 (0.37 vs. 1.67, P<0.05). In addition, we observed that only 22% of patients had recovered ANC >1,500/µL in the infection group as opposed to 70% in the non-infection group at D90 (P<0.05). Fifty-eight percent of the patients (11/19) with relapsed refractory DLBCL achieved a complete response with a median follow-up of 233 days (7.7 months). CONCLUSIONS: Although CAR-T cell therapy is highly effective, infectious complications remain an important cause of morbidity and mortality. Low ANC/ALC and AMC/ALC ratios at D30 are potential novel predictors of infection and can be considered in future prophylactic strategies.

Chronic Immune-Mediated Polyneuropathies.

This article discusses the chronic immune-mediated polyneuropathies, a broad category of acquired polyneuropathies that encompasses chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), the most common immune-mediated neuropathy, the CIDP variants, and the vasculitic neuropathies. Polyneuropathies associated with rheumatological diseases and systemic inflammatory diseases, such as sarcoidosis, will also be briefly covered. These patients' history, examination, serum studies, and electrodiagnostic studies, as well as histopathological findings in the case of vasculitis, confirm the diagnosis and differentiate them from the more common length-dependent polyneuropathies. Prompt identification and initiation of treatment is imperative for these chronic immune-mediated polyneuropathies to prevent disability and even death.

siRNA nanoparticle suppresses drug-resistant gene and prolongs survival in an orthotopic glioblastoma xenograft mouse model.

Temozolomide (TMZ) is the standard of care chemotherapy drug for treating glioblastomas (GBMs), the most aggressive cancer that affects people of all ages. However, its therapeutic efficacy is limited by the drug resistance mediated by a DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), which eliminates the TMZ-induced DNA lesions. Here we report the development of an iron oxide nanoparticle (NP) system for targeted delivery of siRNAs to suppress the TMZ-resistance gene (MGMT). We show that our NP is able to overcome biological barriers, bind specifically to tumor cells, and reduce MGMT expression in tumors of mice bearing orthotopic GBM serially-passaged patient-derived xenografts. The treatment with sequential administration of this NP and TMZ resulted in increased apoptosis of GBM stem-like cells, reduced tumor growth, and significantly-prolonged survival as compared to mice treated with TMZ alone. This study introduces an approach that holds great promise to improve the outcomes of GBM patients.

Iron Oxide Nanoparticles as T1 Contrast Agents for Magnetic Resonance Imaging: Fundamentals, Challenges, Applications, and Prospectives.

Gadolinium-based chelates are a mainstay of contrast agents for magnetic resonance imaging (MRI) in the clinic. However, their toxicity elicits severe side effects and the Food and Drug Administration has issued many warnings about their potential retention in patients' bodies, which causes safety concerns. Iron oxide nanoparticles (IONPs) are a potentially attractive alternative, because of their nontoxic and biodegradable nature. Studies in developing IONPs as T1 contrast agents have generated promising results, but the complex, interrelated parameters influencing contrast enhancement make the development difficult, and IONPs suitable for T1 contrast enhancement have yet to make their way to clinical use. Here, the fundamental principles of MRI contrast agents are discussed, and the current status of MRI contrast agents is reviewed with a focus on the advantages and limitations of current T1 contrast agents and the potential of IONPs to serve as safe and improved alternative to gadolinium-based chelates. The past advances and current challenges in developing IONPs as a T1 contrast agent from a materials science perspective are presented, and how each of the key material properties and environment variables affects the performance of IONPs is assessed. Finally, some potential approaches to develop high-performance and clinically relevant T1 contrast agents are discussed.

Recent Progress in the Synergistic Combination of Nanoparticle-Mediated Hyperthermia and Immunotherapy for Treatment of Cancer.

Immunotherapy has demonstrated great clinical success in certain cancers, driven primarily by immune checkpoint blockade and adoptive cell therapies. Immunotherapy can elicit strong, durable responses in some patients, but others do not respond, and to date immunotherapy has demonstrated success in only a limited number of cancers. To address this limitation, combinatorial approaches with chemo- and radiotherapy have been applied in the clinic. Extensive preclinical evidence suggests that hyperthermia therapy (HT) has considerable potential to augment immunotherapy with minimal toxicity. This progress report will provide a brief overview of immunotherapy and HT approaches and highlight recent progress in the application of nanoparticle (NP)-based HT in combination with immunotherapy. NPs allow for tumor-specific targeting of deep tissue tumors while potentially providing more even heating. NP-based HT increases tumor immunogenicity and tumor permeability, which improves immune cell infiltration and creates an environment more responsive to immunotherapy, particularly in solid tumors.

A highly selective iron oxide-based imaging nanoparticle for long-term monitoring of drug-induced tumor cell apoptosis.

The ability to visualize and quantify apoptosis in vivo is critical to monitoring the disease response to treatment and providing prognostic information. However, the application of current apoptosis labeling probes faces significant challenges including nonspecific tissue uptake, inefficient apoptotic cell labeling and short monitoring windows. Here we report a highly specific apoptosis labeling nanoparticle (NP) probe with Pisum sativum agglutinin (PSA) as a tumor targeting ligand for prolonged in vivo apoptosis imaging. The NP (namely, IONP-Neu-PSA) consists of a magnetic iron oxide core (IONP) conjugated with PSA, and a reporter fluorophore. IONP-Neu-PSA demonstrated minimal cytotoxicity and high labeling specificity towards apoptotic cells in vitro. When applied in vivo, IONP-Neu-PSA tracks apoptotic tumors for a prolonged period of two weeks under near-IR imaging with low background noise. Moreover, IONP-Neu-PSA possesses T2 contrast enhancing properties that can potentially enable apoptosis detection by magnetic resonance imaging (MRI). The high specificity for apoptotic cells, sustained fluorescence signals, and non-invasive imaging capability exhibited by IONP-Neu-PSA make it a versatile tool for cancer treatment monitoring and pathological research.

Microfluidic Synthesis of Iron Oxide Nanoparticles.

Research efforts into the production and application of iron oxide nanoparticles (IONPs) in recent decades have shown IONPs to be promising for a range of biomedical applications. Many synthesis techniques have been developed to produce high-quality IONPs that are safe for in vivo environments while also being able to perform useful biological functions. Among them, coprecipitation is the most commonly used method but has several limitations such as polydisperse IONPs, long synthesis times, and batch-to-batch variations. Recent efforts at addressing these limitations have led to the development of microfluidic devices that can make IONPs of much-improved quality. Here, we review recent advances in the development of microfluidic devices for the synthesis of IONPs by coprecipitation. We discuss the main architectures used in microfluidic device design and highlight the most prominent manufacturing methods and materials used to construct these microfluidic devices. Finally, we discuss the benefits that microfluidics can offer to the coprecipitation synthesis process including the ability to better control various synthesis parameters and produce IONPs with high production rates.

Cocaine analogue conjugated magnetic nanoparticles for labeling and imaging dopaminergic neurons.

Molecular imaging of the dopamine transporter (DAT) with Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) has been widely used in studies of neurological and psychiatric disorders. Nevertheless, there is a great interest in expanding molecular imaging to include magnetic resonance technology, because of the superior spatial resolution this technology may provide. Here we present a magnetic nanoparticle (NP) that specifically targets dopaminergic neurons and allows DAT imaging with magnetic resonance imaging (MRI). The nanoparticle (namely, NP-DN) is composed of an iron oxide core and a polyethylene glycol (PEG) coating to which a DAT specific dopaminergic neurolabeler (DN) is conjugated. NP-DN displayed long-term stability with favorable hydrodynamic size and surface charge suitable for in vivo application. In vitro studies showed NP-DN was non-toxic, displayed specificity towards DAT-expressing neurons, and demonstrated a 3-fold increase in DAT labeling over non-targeted NP. Our study shows NP-DN provides excellent contrast enhancement for MRI and demonstrates great potential for neuroimaging.

In vivo Serum Enabled Production of Ultrafine Nanotherapeutics for Cancer Treatment.

Systemic delivery of hydrophobic anti-cancer drugs with nanocarriers, particularly for drug-resistant and metastatic cancer, remain a challenge because of the difficulty to achieve high drug loading, while maintaining a small hydrodynamic size and colloid stability in blood to ensure delivery of an efficacious amount of drug to tumor cells. Here we introduce a new approach to address this challenge. In this approach, nanofibers of larger size with good drug loading capacity are first constructed by a self-assembly process, and upon intravascular injection and interacting with serum proteins in vivo, these nanofibers break down into ultra-fine nanoparticles of smaller size that inherit the drug loading property from their parent nanofibers. We demonstrate the efficacy of this approach with a clinically available anti-cancer drug: paclitaxel (PTX). In vitro, the PTX-loaded nanoparticles enter cancer cells and induce cellular apoptosis. In vivo, they demonstrate prolonged circulation in blood, induce no systemic toxicity, and show high potency in inhibiting tumor growth and metastasis in both mouse models of aggressive, drug-resistant breast cancer and melanoma. This study points to a new strategy toward improved anti-cancer drug delivery and therapy.

Catalase-Functionalized Iron Oxide Nanoparticles Reverse Hypoxia-Induced Chemotherapeutic Resistance.

Intratumoral hypoxia is a major contributor to multiple drug resistance (MDR) in cancer, and can lead to poor prognosis of patients receiving chemotherapy. Development of an MDR-inhibitor that mitigates the hypoxic environment is crucial for cancer management and treatment. Reported is a biocompatible and biodegradable catalase-conjugated iron oxide nanoparticle (Cat-IONP) capable of converting reactive oxygen species to molecular oxygen to supply an oxygen source for the hypoxic tumor microenvironment. Cat-IONP demonstrates initial enzymatic activity comparable to free catalase while providing a nearly threefold increase in long-term enzymatic activity. It is demonstrated that Cat-IONP significantly reduces the in vitro expression of hypoxia-inducible factors at the transcription level in a breast cancer cell line. Co-treatment of Cat-IONP and paclitaxel (PTX) significantly increases the drug sensitivity of hypoxic-cultured cells, demonstrating greater than twofold and fivefold reduction in cell viability in comparison to cells treated only with 80 and 120 × 10-6 m PTX, respectively. These findings demonstrate the ability of Cat-IONP to act as an MDR-inhibitor at different biological levels, suggesting a promising strategy to combat cancer-MDR and to optimize cancer management and treatment outcomes.

Time-Resolved MRI Assessment of Convection-Enhanced Delivery by Targeted and Nontargeted Nanoparticles in a Human Glioblastoma Mouse Model.

Convection-enhanced delivery (CED) provides direct access of infusates to brain tumors; however, clinical translation of this technology has not been realized because of the inability to accurately visualize infusates in real-time and lack of targeting modalities against diffuse cancer cells. In this study, we use time-resolved MRI to reveal the kinetics of CED processes in a glioblastoma (GBM) model using iron oxide nanoparticles (NP) modified with a glioma-targeting ligand, chlorotoxin (CTX). Mice bearing orthotopic human GBM tumors were administered a single dose of targeted CTX-conjugated NP (NPCP-CTX) or nontargeted NP (NPCP) via CED. High-resolution T2-weighted, T2*-weighted, and quantitative T2 MRI were utilized to image NP delivery in real time and determined the volume of distribution (VD) of NPs at multiple time points over the first 48 hours post-CED. GBM-specific targeting was evaluated by flow cytometry and intracellular NP localization by histologic assessment. NPCP-CTX produced a VD of 121 ± 39 mm3 at 24 hours, a significant increase compared with NPCP, while exhibiting GBM specificity and localization to cell nuclei. Notably, CED of NPCP-CTX resulted in a sustained expansion of VD well after infusion, suggesting a possible active transport mechanism, which was further supported by the presence of NPs in endothelial and red blood cells. In summary, we show that time-resolved MRI is a suitable modality to study CED kinetics, and CTX-mediated CED facilitates extensive distribution of infusate and specific targeting of tumor cells. SIGNIFICANCE: MRI is used to monitor convection-enhanced delivery in real time using a nanoparticle-based contrast agent, and glioma-specific targeting significantly improves the volume of distribution in tumors.

Theranostic Nanoparticles for RNA-Based Cancer Treatment.

Certain genetic mutations lead to the development of cancer through unchecked cell growth and division. Cancer is typically treated through surgical resection, radiotherapy, and small-molecule chemotherapy. A relatively recent approach to cancer therapy involves the use of a natural process wherein small RNA molecules regulate gene expression in a pathway known as RNA interference (RNAi). RNA oligomers pair with a network of proteins to form an RNA-induced silencing complex, which inhibits the translation of mRNA into proteins, thereby controlling the expression of gene products. Synthetically produced RNA oligomers may be designed to target and silence specific oncogenes to provide cancer therapy. The primary challenges facing the use of the RNAi pathway for cancer therapy are the safe and efficacious delivery of RNA payloads and their release at pertinent sites within disease-causing cells. Nucleases are abundant in the bloodstream and intracellular environment, and therapeutic RNA sequences often require a suitable carrier to provide protection from degradation prior to reaching their site of action in the body. The use of metal core nanoparticles (NPs) serving as targeted delivery vehicles able to shield and direct RNA payloads to their intended destinations have recently gained favor. Biological barriers present in the body establish a size prerequisite for drug delivery vehicles; to overcome recognition by the body's immune system and to gain access to intracellular environments, drug carriers must be small (< 100 nm). Iron oxide and gold core NPs can be synthesized with a high degree of control to create uniform ultrasmall drug delivery vehicles capable of bypassing key biological barriers. While progress is being made in size control of liposomal and polymer NPs, such advances still lag in comparison to the exquisite tunability and time stability of size engineering achievable with metal core NPs at bulk scales. Further, unlike lipid- and viral-based transfection agents, the biodistribution of metal core NPs can be traced using noninvasive imaging techniques that capitalize on the interaction of electromagnetic radiation and the inorganic atoms at the core of the NPs. Finally, metal core NPs have been shown to match the transfection efficiency of conventional RNA-delivery vehicles while also providing less immunogenicity and minimal side effects through the addition of tumor-targeting ligands on their surface. This Account reviews recent advances in the use of iron oxide and gold NPs for RNAi therapy. An overview of the different types of RNA-based therapies is provided along with a discussion of the advantages and current limitations of the technique. We highlight design considerations for the use of iron oxide and gold NP carriers in RNAi, including a discussion of the importance of size and its role in traversing biological barriers, NP surface modifications required for targeted delivery and RNA payload release, and auxiliary properties supporting imaging functionality for treatment monitoring. Applications of NPs for combination therapies including the pairing of RNAi with chemotherapy, photothermal therapy, immunotherapy, and radiotherapy are explored through examples. Finally, future perspectives are provided with a focus on the current limitations and the potential for clinical translation of iron oxide and gold NPs in RNAi therapy.

Biconcave Carbon Nanodisks for Enhanced Drug Accumulation and Chemo-Photothermal Tumor Therapy.

It is considered a significant challenge to construct nanocarriers that have high drug loading capacity and can overcome physiological barriers to deliver efficacious amounts of drugs to solid tumors. Here, the development of a safe, biconcave carbon nanodisk to address this challenge for treating breast cancer is reported. The nanodisk demonstrates fluorescent imaging capability, an exceedingly high loading capacity (947.8 mg g-1 , 94.78 wt%) for doxorubicin (DOX), and pH-responsive drug release. It exhibits a higher uptake rate by tumor cells and greater accumulation in tumors in a mouse model than its carbon nanosphere counterpart. In addition, the nanodisk absorbs and transforms near-infrared (NIR) light to heat, which enables simultaneous NIR-responsive drug release for chemotherapy and generation of thermal energy for tumor cell destruction. Notably, this NIR-activated dual therapy demonstrates a near complete suppression of tumor growth in a mouse model of triple-negative breast cancer when DOX-loaded nanodisks are administered systemically.

Nanoparticle Biokinetics in Mice and Nonhuman Primates.

Despite the preponderance of iron oxide nanoparticles (NPs) designed for theranostic applications, widespread clinical translation of these NPs lags behind. A better understanding of how NP pharmacokinetics vary between small and large animal models is needed to rapidly customize NPs for optimal performance in humans. Here we use noninvasive magnetic resonance imaging (MRI) to track iron oxide NPs through a large number of organ systems in vivo to investigate NP biokinetics in both mice and nonhuman primates. We demonstrate that pharmacokinetics are similar between mice and macaques in the blood, liver, spleen, and muscle, but differ in the kidneys, brain, and bone marrow. Our study also demonstrates that full-body MRI is practical, rapid, and cost-effective for tracking NPs noninvasively with high spatiotemporal resolution. Our techniques using a nonhuman primate model may provide a platform for testing a range of NP formulations.

Mesoporous carbon nanoshells for high hydrophobic drug loading, multimodal optical imaging, controlled drug release, and synergistic therapy.

Loading and controlled release of sufficient hydrophobic drugs to tumor cells has been the bottleneck in chemotherapy for decades. Herein we report the development of a fluorescent and mesoporous carbon nanoshell (FMP-CNS) that exhibits a loading capacity for the hydrophobic drug paclitaxel (PTX) as high as ∼80 wt% and releases the drug in a controllable fashion under NIR irradiation (825 nm) at an intensity of 1.5 W cm-2. The high drug loading is primarily attributed to its mesoporous structure and to the supramolecular π-stacking between FMP-CNSs and PTX molecules. The FMP-CNS also exhibits wavelength-tunable and upconverted fluorescence properties and thus can serve as an optical marker for confocal, two-photon, and near infrared (NIR) fluorescence imaging. Furthermore, our in vitro results indicate that FMP-CNSs demonstrate high therapeutic efficacy through the synergistic effect of combined chemo-photothermal treatment. In vivo studies demonstrate marked suppression of tumor growth in mice bearing rat C6 glioblastoma after administration with a single intratumoral injection of PTX-loaded FMP-CNS.

pH-Sensitive O6-Benzylguanosine Polymer Modified Magnetic Nanoparticles for Treatment of Glioblastomas.

Nanoparticle-mediated delivery of chemotherapeutics has demonstrated potential in improving anticancer efficacy by increasing serum half-life and providing tissue specificity and controlled drug release to improve biodistribution of hydrophobic chemotherapeutics. However, suboptimal drug loading, particularly for solid core nanoparticles (NPs), remains a challenge that limits their clinical application. In this study we formulated a NP coated with a pH-sensitive polymer of O6-methylguanine-DNA methyltransferase (MGMT) inhibitor analog, dialdehyde modified O6-benzylguanosine (DABGS) to achieve high drug loading, and polyethylene glycol (PEG) to ameliorate water solubility and maintain NP stability. The base nanovector consists of an iron oxide core (9 nm) coated with hydrazide functionalized PEG (IOPH). DABGS and PEG-dihydrazide were polymerized on the iron oxide nanoparticle surface (IOPH-pBGS) through acid-labile hydrazone bonds utilizing a rapid, freeze-thaw catalysis approach. DABGS polymerization was confirmed by FTIR and quantitated by UV-vis spectroscopy. IOPH-pBGS demonstrated excellent drug loading of 33.4 ± 5.1% by weight while maintaining small size (36.5 ± 1.8 nm). Drug release was monitored at biologically relevant pHs and demonstrated pH dependent release with maximum release at pH 5.5 (intracellular conditions), and minimal release at physiological pH (7.4). IOPH-pBGS significantly suppressed activity of MGMT and potentiated Temozolomide (TMZ) toxicity in vitro, demonstrating potential as a new treatment option for glioblastomas (GBMs).

Approach to Rapid Synthesis and Functionalization of Iron Oxide Nanoparticles for High Gene Transfection.

Surface functionalization of theranostic nanoparticles (NPs) typically relies on lengthy, aqueous postsynthesis labeling chemistries that have limited ability to fine-tune surface properties and can lead to NP heterogeneity. The need for a rapid, simple synthesis approach that can provide great control over the display of functional moieties on NP surfaces has led to increased use of highly selective bioorthoganol chemistries including metal-affinity coordination. Here we report a simple approach for rapid production of a superparamagnetic iron oxide NPs (SPIONs) with tunable functionality and high reproducibility under aqueous conditions. We utilize the high affinity complex formed between catechol and Fe((III)) as a means to dock well-defined catechol modified polymer modules on the surface of SPIONs during sonochemical coprecipitation synthesis. Polymer modules consisted of chitosan and poly(ethylene glycol) (PEG) copolymer (CP) modified with catechol (CCP), and CCP functionalized with cationic polyethylenimine (CCP-PEI) to facilitate binding and delivery of DNA for gene therapy. This rapid synthesis/functionalization approach provided excellent control over the extent of PEI labeling, improved SPION magnetic resonance imaging (MRI) contrast enhancement and produced an efficient transfection agent.

Iron-Oxide-Based Nanovector for Tumor Targeted siRNA Delivery in an Orthotopic Hepatocellular Carcinoma Xenograft Mouse Model.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Small interfering RNA (siRNA) holds promise as a new class of therapeutics for HCC, as it can achieve sequence-specific gene knockdown with low cytotoxicity. However, the main challenge in the clinical application of siRNA lies in the lack of effective delivery approaches that need to be highly specific and thus incur low or no systemic toxicity. Here, a nonviral nanoparticle-based gene carrier is presented that can specifically deliver siRNA to HCC. The nanovector (NP-siRNA-GPC3 Ab) is made of an iron oxide core coated with chitosan-polyethylene glycol (PEG) grafted polyethyleneimine copolymer, which is further functionalized with siRNA and conjugated with a monoclonal antibody (Ab) against human glypican-3 (GPC3) receptor highly expressed in HCC. A rat RH7777 HCC cell line that coexpresses human GPC3 and firefly luciferase (Luc) is established to evaluate the nanovector. The nanoparticle-mediated delivery of siRNA against Luc effectively suppresses Luc expression in vitro without notable cytotoxicity. Significantly, NP-siLuc-GPC3 Ab administered intravenously in an orthotopic model of HCC is able to specifically bound to tumor and induce remarkable inhibition of Luc expression. The findings demonstrate the potential of using this nanovector for targeted delivery of therapeutic siRNA to HCC.

3D Porous Chitosan-Alginate Scaffolds as an In Vitro Model for Evaluating Nanoparticle-Mediated Tumor Targeting and Gene Delivery to Prostate Cancer.

Cationic nanoparticles (NPs) for targeted gene delivery are conventionally evaluated using 2D in vitro cultures. However, this does not translate well to corresponding in vivo studies because of the marked difference in NP behavior in the presence of the tumor microenvironment. In this study, we investigated whether prostate cancer (PCa) cells cultured in three-dimensional (3D) chitosan-alginate (CA) porous scaffolds could model cationic NP-mediated gene targeted delivery to tumors in vitro. We assessed in vitro tumor cell proliferation, formation of tumor spheroids, and expression of marker genes that promote tumor malignancy in CA scaffolds. The efficacy of NP-targeted gene delivery was evaluated in PCa cells in 2D cultures, PCa tumor spheroids grown in CA scaffolds, and PCa tumors in a mouse TRAMP-C2 flank tumor model. PCa cells cultured in CA scaffolds grew into tumor spheroids and displayed characteristics of higher malignancy as compared to those in 2D cultures. Significantly, targeted gene delivery was only observed in cells cultured in CA scaffolds, whereas cells cultured on 2D plates showed no difference in gene delivery between targeted and nontarget control NPs. In vivo NP evaluation confirmed targeted gene delivery, indicating that only CA scaffolds correctly modeled NP-mediated targeted delivery in vivo. These findings suggest that CA scaffolds serve as a better in vitro platform than 2D cultures for evaluation of NP-mediated targeted gene delivery to PCa.