Hyaluronic Acid Nanoparticles for Immunogenic Chemotherapy of Leukemia and T-Cell Lymphoma.

Ratiometric delivery of combination chemotherapy can achieve therapeutic efficacy based on synergistic interactions between drugs. It is critical to design such combinations with drugs that complement each other and reduce cancer growth through multiple mechanisms. Using hyaluronic acid (HA) as a carrier, two chemotherapeutic agents-doxorubicin (DOX) and camptothecin (CPT)-were incorporated and tested for their synergistic potency against a broad panel of blood-cancer cell lines. The pair also demonstrated the ability to achieve immunogenic cell death by increasing the surface exposure levels of Calreticulin, thereby highlighting its ability to induce apoptosis via an alternate pathway. Global proteomic profiling of cancer cells treated with HA-DOX-CPT identified pathways that could potentially predict patient sensitivity to HA-DOX-CPT. This lays the foundation for further exploration of integrating drug delivery and proteomics in personalized immunogenic chemotherapy.

Hyaluronic acid-doxorubicin nanoparticles for targeted treatment of colorectal cancer.

Colorectal cancer, common in both men and women, occurs when tumors form in the linings of the colon. Common treatments of colorectal cancer include surgery, chemotherapy, and radiation therapy; however, many colorectal cancer treatments often damage healthy tissues and cells, inducing severe side effects. Conventional chemotherapeutic agents such as doxorubicin (Dox) can be potentially used for the treatment of colorectal cancer; however, they suffer from limited targeting and lack of selectivity. Here, we report that doxorubicin complexed to hyaluronic acid (HA) (HA-Dox) exhibits an unusual behavior of high accumulation in the intestines for at least 24 hr when injected intravenously. Intravenous administrations of HA-Dox effectively preserved the mucosal epithelial intestinal integrity in a chemical induced colon cancer model in mice. Moreover, treatment with HA-Dox decreased the expression of intestinal apoptotic and inflammatory markers. The results suggest that HA-Dox could effectively inhibit the development of colorectal cancer in a safe manner, which potentially be used a promising therapeutic option.

Gemcitabine and doxorubicin in immunostimulatory monophosphoryl lipid A liposomes for treating breast cancer.

Cancer therapy is increasingly shifting toward targeting the tumor immune microenvironment and influencing populations of tumor infiltrating lymphocytes. Breast cancer presents a unique challenge as tumors of the triple-negative breast cancer subtype employ a multitude of immunosilencing mechanisms that promote immune evasion and rapid growth. Treatment of breast cancer with chemotherapeutics has been shown to induce underlying immunostimulatory responses that can be further amplified with the addition of immune-modulating agents. Here, we investigate the effects of combining doxorubicin (DOX) and gemcitabine (GEM), two commonly used chemotherapeutics, with monophosphoryl lipid A (MPLA), a clinically used TLR4 adjuvant derived from liposaccharides. MPLA was incorporated into the lipid bilayer of liposomes loaded with a 1:1 molar ratio of DOX and GEM to create an intravenously administered treatment. In vivo studies indicated excellent efficacy of both GEM-DOX liposomes and GEM-DOX-MPLA liposomes against 4T1 tumors. In vitro and in vivo results showed increased dendritic cell expression of CD86 in the presence of liposomes containing chemotherapeutics and MPLA. Despite this, a tumor rechallenge study indicated little effect on tumor growth upon rechallenge, indicating the lack of a long-term immune response. GEM/DOX/MPLA-L displayed remarkable control of the primary tumor growth and can be further explored for the treatment of triple-negative breast cancer with other forms of immunotherapy.

Optimized 5-Fluorouridine Prodrug for Co-Loading with Doxorubicin in Clinically Relevant Liposomes.

Liposome-based drug delivery systems have allowed for better drug tolerability and longer circulation times but are often optimized for a single agent due to the inherent difficulty of co-encapsulating two drugs with differing chemical profiles. Here, we design and test a prodrug based on a ribosylated nucleoside form of 5-fluorouracil, 5-fluorouridine (5FUR), with the final purpose of co-encapsulation with doxorubicin (DOX) in liposomes. To improve the loading of 5FUR, we developed two 5FUR prodrugs that involved the conjugation of either one or three moieties of tryptophan (W) known respectively as, 5FUR-W and 5FUR-W3. 5FUR-W demonstrated greater chemical stability than 5FUR-W3 and allowed for improved loading with fewer possible byproducts from tryptophan hydrolysis. Varied drug ratios of 5FUR-W: DOX were encapsulated for in vivo testing in the highly aggressive 4T1 murine breast cancer model. A liposomal molar ratio of 2.5 5FUR-W: DOX achieved a 62.6% reduction in tumor size compared to the untreated control group and a 33% reduction compared to clinical doxorubicin liposomes in a proof-of-concept study to demonstrate the viability of the co-encapsulated liposomes. We believe that the new prodrug 5FUR-W demonstrates a prodrug design with clinical translatability by reducing the number of byproducts produced by the hydrolysis of tryptophan, while also allowing for loading flexibility.

Enhancement of Anticancer Efficacy and Tumor Penetration of Sorafenib by Ionic Liquids.

Ionic liquids (ILs) possess unique solvation and biological properties for drug delivery. Choline and geranic acid (CAGE) in particular, has been successfully formulated to orally deliver insulin and hydrophobic therapeutics such as sorafenib (SRF). However, relatively little is known about the effect of CAGE on intracellular delivery of drugs. Here the effect of low-concentration CAGE (<2 mg mL-1 ) on the delivery of SRF into cancer cells (4T1, PANC-1, and HT29) as well as intestine epithelium cells (Caco-2) is studied. The anti-cancer effect of SRF is enhanced by up to fivefold in the presence of CAGE (0.5 mg mL-1 ). The effect is mediated not by enhancing the cellular uptake of SRF but by improving intracellular SRF retention by inhibiting exocytosis. Moreover, CAGE improves the anti-tumor effect of SRF by increasing apoptosis and blocking cell-cycle progression. Moreover, CAGE significantly enhances the penetration of SRF into and across multicellular constructs with multiple mechanisms involved. Collectively, the administration of ILs such as CAGE combined with SRF may offer a novel therapy to better inhibit tumor progression.

Engineering of Living Cells with Polyphenol-Functionalized Biologically Active Nanocomplexes.

Approaches to safely and effectively augment cellular functions without compromising the inherent biological properties of the cells, especially through the integration of biologically labile domains, remain of great interest. Here, a versatile strategy to assemble biologically active nanocomplexes, including proteins, DNA, mRNA, and even viral carriers, on cellular surfaces to generate a cell-based hybrid system referred to as "Cellnex" is established. This strategy can be used to engineer a wide range of cell types used in adoptive cell transfers, including erythrocytes, macrophages, NK cells, T cells, etc. Erythrocytenex can enhance the delivery of cargo proteins to the lungs in vivo by 11-fold as compared to the free cargo counterpart. Biomimetic microfluidic experiments and modeling provided detailed insights into the targeting mechanism. In addition, Macrophagenex is capable of enhancing the therapeutic efficiency of anti-PD-L1 checkpoint inhibitors in vivo. This simple and adaptable approach may offer a platform for the rapid generation of complex cellular systems.

Cellular backpacks for macrophage immunotherapy.

Adoptive cell transfers have emerged as a disruptive approach to treat disease in a manner that is more specific than using small-molecule drugs; however, unlike traditional drugs, cells are living entities that can alter their function in response to environmental cues. In the present study, we report an engineered particle referred to as a "backpack" that can robustly adhere to macrophage surfaces and regulate cellular phenotypes in vivo. Backpacks evade phagocytosis for several days and release cytokines to continuously guide the polarization of macrophages toward antitumor phenotypes. We demonstrate that these antitumor phenotypes are durable, even in the strongly immunosuppressive environment of a murine breast cancer model. Conserved phenotypes led to reduced metastatic burdens and slowed tumor growths compared with those of mice treated with an equal dose of macrophages with free cytokine. Overall, these studies highlight a new pathway to control and maintain phenotypes of adoptive cellular immunotherapies.

Design principles of drug combinations for chemotherapy.

Combination chemotherapy is the leading clinical option for cancer treatment. The current approach to designing drug combinations includes in vitro optimization to maximize drug cytotoxicity and/or synergistic drug interactions. However, in vivo translatability of drug combinations is complicated by the disparities in drug pharmacokinetics and activity. In vitro cellular assays also fail to represent the immune response that can be amplified by chemotherapy when dosed appropriately. Using three common chemotherapeutic drugs, gemcitabine (GEM), irinotecan (IRIN), and a prodrug form of 5-flurouracil (5FURW), paired with another common drug and immunogenic cell death inducing agent, doxorubicin (DOX), we sought to determine the in vitro parameters that predict the in vivo outcomes of drug combinations in the highly aggressive orthotopic 4T1 murine breast cancer model. With liposomal encapsulation of each drug pair, we enabled uniform drug pharmacokinetics across the drug combinations, thus allowing us to study the inherent benefits of the drug pairs and compare them to DOX liposomes representative of DOXIL®. Surprisingly, the Hill coefficient (HC) of the in vitro dose-response Hill equation provided a better prediction of in vivo efficacy than drug IC50 or combination index. GEM/DOX liposomes exhibited a high HC in vitro and an increase in M1/M2 macrophage ratio in vivo. Hence, GEM/DOX liposomes were further investigated in a long-term survival study and compared against doxorubicin liposomes and gemcitabine liposomes. The GEM/DOX liposome-treated group had the longest median survival time, double that of the DOX liposome-treated group and 3.4-fold greater than that of the untreated controls. Our studies outline the development of a more efficacious formulation than clinically representative liposomal doxorubicin for breast cancer treatment and presents a novel strategy for designing cancer drug combinations.

Effect of Nanoparticle Composition, Size, Shape, and Stiffness on Penetration Across the Blood-Brain Barrier.

The delivery of therapeutics to the brain in an efficient, noninvasive manner continues to be a major unmet need in the field of drug delivery. One significant impediment to brain delivery results from the existence of the physical yet dynamic blood-brain barrier (BBB). Despite the many, often complex strategies that currently exist to breach the BBB, adequate delivery of effective therapeutics from the bloodstream continues to remain quite low. Nanotechnology has emerged as a promising tool for brain delivery, but little is known about the important particle parameters that influence delivery. Here, we synthesized and characterized a library of nanoparticles with distinct properties ranging from size, shape, stiffness, and composition to investigate and identify the key attributes influencing particle uptake and transport for brain delivery. To accomplish this task, an in vitro human BBB model was developed and validated using human cerebral microvascular endothelial cells (hCMEC/D3). Particle uptake and apparent permeability coefficients (Papp) were then determined for each particle group. To elucidate the roles of different parameters on particle uptake and transport across the BBB, the predominant mechanisms of endocytosis were also investigated. Our results show that particle composition yielded the greatest impact on penetration across the BBB model. This work lays the foundation and provides new insights into the role of particle parameters on penetration across the BBB.

Role of synergy and immunostimulation in design of chemotherapy combinations: An analysis of doxorubicin and camptothecin.

Combination chemotherapy is often employed to improve therapeutic efficacies of drugs. However, traditional combination regimens often utilize drugs at or near-their maximum tolerated doses (MTDs), elevating the risk of dose-related toxicity and impeding their clinical success. Further, high doses of adjuvant or neoadjuvant chemotherapies can cause myeloablation, which compromises the immune response and hinders the efficacy of chemotherapy as well as accompanying treatments such as immunotherapy. Clinical outcomes can be improved if chemotherapy combinations are designed to reduce the overall doses without compromising their therapeutic efficacy. To this end, we investigated a combination of camptothecin (CPT) with doxorubicin (DOX) as a low-dose treatment option for breast cancer. DOX-CPT combinations were synergistic in several breast cancer cell lines in vitro and one particular ratio displayed extremely high synergy on human triple negative breast cancer cells (MDA-MB-231). This combination led to excellent long-term survival of mice bearing MDA-MB-231 tumors at doses roughly five-fold lower than the reported MTD values of its constituent drugs. Impact of low dose DOX-CPT treatment on local tumor immune environment was assessed in immunocompetent mice bearing breast cancer (4T1) tumors. The combination was not only superior in inhibiting the disease progression compared to individual drugs, but it also generated a more favorable antitumor immunogenic response. Engineering DOX and CPT ratios to manifest synergy enables treatment at doses much lower than their MTDs, which could ultimately facilitate their translation into the clinic as a promising combination for breast cancer treatment.

Immunological consequences of chemotherapy: Single drugs, combination therapies and nanoparticle-based treatments.

The tumor environment has been shown to employ several immunosuppressive mechanisms to evade cancer treatments. While immunotherapies actively reverse such mechanisms and polarize the immune system against malignant cells, combining immunotherapy with certain chemotherapeutics can lead to increased efficacy compared to either treatment alone. Low-dose chemotherapy demonstrates several immunogenic effects that can favorably potentiate immunotherapies. However, the clinical benefits of such therapies are confounded by treatment complexity and marginal improvements. The highly complex relationship between chemotherapeutic drug dosing and subsequent immunological consequences is often generalized, thus limiting their efficacy and potential. Also, continuous monitoring of the immunological impact is crucial for designing superior synergies while optimizing chemotherapeutic combinations or chemotherapeutics in novel delivery systems. In this review, we summarize the existing literature on the immunological outcomes of chemotherapies administered individually, in combination regimens, and in formulation with novel delivery agents. Further, we discuss the relevance of key parameters including dosage, schedule, and tumor models, and describe their clinical implications with an emphasis on approaches and evaluations that are crucial for developing effective immune-stimulating therapies.