Advances in Nanomedicine Design: Multidisciplinary Strategies for Unmet Medical Needs.

Globally, a rising burden of complex diseases takes a heavy toll on human lives and poses substantial clinical and economic challenges. This review covers nanomedicine and nanotechnology-enabled advanced drug delivery systems (DDS) designed to address various unmet medical needs. Key nanomedicine and DDSs, currently employed in the clinic to tackle some of these diseases, are discussed focusing on their versatility in diagnostics, anticancer therapy, and diabetes management. First-hand experiences from our own laboratory and the work of others are presented to provide insights into strategies to design and optimize nanomedicine- and nanotechnology-enabled DDS for enhancing therapeutic outcomes. Computational analysis is also briefly reviewed as a technology for rational design of controlled release DDS. Further explorations of DDS have illuminated the interplay of physiological barriers and their impact on DDS. It is demonstrated how such delivery systems can overcome these barriers for enhanced therapeutic efficacy and how new perspectives of next-generation DDS can be applied clinically.

Optimizing the Design of Blood-Brain Barrier-Penetrating Polymer-Lipid-Hybrid Nanoparticles for Delivering Anticancer Drugs to Glioblastoma.

PURPOSE: Chemotherapy for glioblastoma multiforme (GBM) remains ineffective due to insufficient penetration of therapeutic agents across the blood-brain barrier (BBB) and into the GBM tumor. Herein, is described, the optimization of the lipid composition and fabrication conditions for a BBB- and tumor penetrating terpolymer-lipid-hybrid nanoparticle (TPLN) for delivering doxorubicin (DOX) to GBM. METHODS: The composition of TPLNs was first screened using different lipids based on nanoparticle properties and in vitro cytotoxicity by using 23 full factorial experimental design. The leading DOX loaded TPLNs (DOX-TPLN) were prepared by further optimization of conditions and used to study cellular uptake mechanisms, in vitro cytotoxicity, three-dimensional (3D) glioma spheroid penetration, and in vivo biodistribution in a murine orthotopic GBM model. RESULTS: Among various lipids studied, ethyl arachidate (EA) was found to provide excellent nanoparticle properties e.g., size, polydispersity index (PDI), zeta potential, encapsulation efficiency, drug loading, and colloidal stability, and highest anticancer efficacy for DOX-TPLN. Further optimized EA-based TPLNs were prepared with an optimal particle size (103.8 ± 33.4 nm) and PDI (0.208 ± 0.02). The resultant DOX-TPLNs showed ~ sevenfold higher efficacy than free DOX against human GBM U87-MG-RED-FLuc cells in vitro. The interaction between the TPLNs and the low-density lipoprotein receptors also facilitated cellular uptake, deep penetration into 3D glioma spheroids, and accumulation into the in vivo brain tumor regions of DOX-TPLNs. CONCLUSION: This work demonstrated that the TPLN system can be optimized by rational selection of lipid type, lipid content, and preparation conditions to obtain DOX-TPLN with enhanced anticancer efficacy and GBM penetration and accumulation.

Importance of integrating nanotechnology with pharmacology and physiology for innovative drug delivery and therapy - an illustration with firsthand examples.

Nanotechnology has been applied extensively in drug delivery to improve the therapeutic outcomes of various diseases. Tremendous efforts have been focused on the development of novel nanoparticles and delineation of the physicochemical properties of nanoparticles in relation to their biological fate and functions. However, in the design and evaluation of these nanotechnology-based drug delivery systems, the pharmacology of delivered drugs and the (patho-)physiology of the host have received less attention. In this review, we discuss important pharmacological mechanisms, physiological characteristics, and pathological factors that have been integrated into the design of nanotechnology-enabled drug delivery systems and therapies. Firsthand examples are presented to illustrate the principles and advantages of such integrative design strategies for cancer treatment by exploiting 1) intracellular synergistic interactions of drug-drug and drug-nanomaterial combinations to overcome multidrug-resistant cancer, 2) the blood flow direction of the circulatory system to maximize drug delivery to the tumor neovasculature and cells overexpressing integrin receptors for lung metastases, 3) endogenous lipoproteins to decorate nanocarriers and transport them across the blood-brain barrier for brain metastases, and 4) distinct pathological factors in the tumor microenvironment to develop pH- and oxidative stress-responsive hybrid manganese dioxide nanoparticles for enhanced radiotherapy. Regarding the application in diabetes management, a nanotechnology-enabled closed-loop insulin delivery system was devised to provide dynamic insulin release at a physiologically relevant time scale and glucose levels. These examples, together with other research results, suggest that utilization of the interplay of pharmacology, (patho-)physiology and nanotechnology is a facile approach to develop innovative drug delivery systems and therapies with high efficiency and translational potential.

Polymer-lipid hybrid nanoparticles synchronize pharmacokinetics of co-encapsulated doxorubicin-mitomycin C and enable their spatiotemporal co-delivery and local bioavailability in breast tumor.

UNLABELLED: Effective combination chemotherapy requires the delivery of drugs of synergism to tumor sites while sparing normal tissues. Herein we investigated whether coencapsulation of doxorubicin and mitomycin C within polymer-lipid hybrid nanoparticles (DMPLN) achieved this goal via ratiometric drugs in an orthotopic murine breast tumor model with nanocarrier-modified biodistribution, pharmacokinetics, local bioavailability and toxicity. Fluorescence imaging revealed quickened and extended tumor uptake but reduced cardiac accumulation of DMPLN. Quantitative drug analysis demonstrated prolonged systemic circulation, increased tumor accumulation and sustained synergistic ratios of doxorubicin and mitomycin C delivered by DMPLN over 24h. Higher levels of tumor cell apoptosis and reduced organ toxicity were obtained with DMPLN compared to free drug cocktails. DMPLN released DOX in tumors more efficiently than that from liposomal doxorubicin, as evidenced by a higher extent of the metabolite, doxorubicinol. These findings substantiate the importance of rational design of nanoparticles for synergistic drug combination therapy. FROM THE CLINICAL EDITOR: The treatment of cancer usually involves using combination chemotherapeutic agents. In adopting a nanomedicine approach, one can in theory design combination therapy consisting of drugs of synergistic activities, with the aim to target tumor specifically while minimizing systemic toxicity. The authors in this study provided evidence for this rational design by co-encapsulation of doxorubicin and mitomycin C within polymer-lipid hybrid nanoparticles (DMPLN) in a breast cancer model.

Synergistic nanoparticulate drug combination overcomes multidrug resistance, increases efficacy, and reduces cardiotoxicity in a nonimmunocompromised breast tumor model.

Anthracyclines, commonly employed for cancer chemotherapy, suffer from dose-limiting cardiotoxicity and poor efficacy due to multidrug resistance (MDR). We previously demonstrated that simultaneous delivery of the synergistic drugs doxorubicin (DOX) and mitomycin C (MMC) by polymer-lipid hybrid nanoparticles (PLN) circumvented MDR, increased efficacy, and reduced cardiotoxicity in immuncompromised mice superior to poly(ethylene glycol)-coated (PEGylated) lipososmal DOX (PLD). Herein it is shown that the DOX-MMC combination was also synergistic in MDR EMT6/AR1 murine breast cancer cells and that their nanoparticle formulations were able to overcome the MDR phenotype. In contrast PLD exhibited little or no effect on the MDR cells. For the first time, these differences in in vitro efficacy are shown to be strongly correlated with cellular uptake and intracellular distribution of DOX brought about by DOX formulations (e.g., free solution, PLN vs PLD). To take into consideration the role of an intact immune system and tumor stroma in the response of host and tumor to chemotherapy, use was made of nonimmunocomprised mouse models to study the dose tolerance, cardiotoxicity, and efficacy of DOX-MMC coloaded PLN (DMsPLN) compared to PLD. DMsPLN treatment at 50 mg/m(2) DOX and 17 mg/m(2) of MMC singly or once every 4 days for 4 cycles were well tolerated by the mice without elevated systemic toxicity blood markers or myocardial damage. In contrast, PLD was limited to a single treatment due to significant total weight loss. The DMsPLN treatment delayed tumor growth up to 312% and 28% in EMT6/WT and EMT6/AR1 models, respectively. This work supports the translational value of DMsPLN for the aggressive management of either naïve or anthracycline-resistant tumors.

Evaluation of the biodistribution and preliminary safety profile of a novel brain-targeted manganese dioxide-based nanotheranostic system for Alzheimer's disease.

A novel brain-targeted and reactive oxygen species-activatable manganese dioxide containing nanoparticle system functionalized with anti-amyloid-ß antibody (named aAß-BTRA-NC) developed by our group has shown great promise as a highly selective magnetic resonance imaging (MRI) contrast agent for early detection and multitargeted disease-modifying treatment of Alzheimer's disease (AD). To further evaluate the suitability of the formulation for future clinical application, we investigated the safety, biodistribution, and pharmacokinetic profile of aAß-BTRA-NC in a transgenic TgCRND8 mouse AD model, wild type (WT) littermate, and CD-1 mice. Dose-ascending studies demonstrated that aAß-BTRA-NC was well-tolerated by the animals up to 300 µmol Mn/kg body weight [b.w.], 3 times the efficacious dose for early AD detection without apparent adverse effects; Histopathological, hematological, and biochemical analyses indicated that a single dose of aAß-BTRA-NC did not cause any toxicity in major organs. Immunotoxicity data showed that aAß-BTRA-NC was safer than commercially available gadolinium-based MRI contrast agents at an equivalent dose of 100 µmol/kg b.w. of metal ions. Intravenously administered aAß-BTRA-NC was taken up by main organs with the order of liver, kidneys, intestines, spleen, followed by other organs, and cleared after one day to one week post injection. Pharmacokinetic analysis indicated that the plasma concentration profile of aAß-BTRA-NC followed a 2-compartmental model with faster clearance in the AD mice than in the WT mice. The results suggest that aAß-BTRA-NC exhibits a strong safety profile as a nanotheranostic agent which warrants more robust preclinical development for future clinical applications.

Biocompatible and bioactivable terpolymer-lipid-MnO2 Nanoparticle-based MRI contrast agent for improving tumor detection and delineation.

Early and precise detection of solid tumor cancers is critical for improving therapeutic outcomes. In this regard, magnetic resonance imaging (MRI) has become a useful tool for tumor diagnosis and image-guided therapy. However, its effectiveness is limited by the shortcomings of clinically available gadolinium-based contrast agents (GBCAs), i.e. poor tumor penetration and retention, and safety concerns. Thus, we have developed a novel nanoparticulate contrast agent using a biocompatible terpolymer and lipids to encapsulate manganese dioxide nanoparticles (TPL-MDNP). The TPL-MDNP accumulated in tumor tissue and produced paramagnetic Mn2+ ions, enhancing T1-weight MRI contrast via the reaction with H2O2 rich in the acidic tumor microenvironment. Compared to the clinically used GBCA, Gadovist®1.0, TPL-MDNP generated stronger T1-weighted MR signals by over 2.0-fold at 30 % less of the recommended clinical dose with well-defined tumor delineation in preclinical orthotopic tumor models of brain, breast, prostate, and pancreas. Importantly, the MRI signals were retained for 60 min by TPL-MDNP, much longer than Gadovist®1.0. Biocompatibility of TPL-MDNP was evaluated and found to be safe up to 4-fold of the dose used for MRI. A robust large-scale manufacturing process was developed with batch-to-batch consistency. A lyophilization formulation was designed to maintain the nanostructure and storage stability of the new contrast agent.

Matrigel alters the pathophysiology of orthotopic human breast adenocarcinoma xenografts with implications for nanomedicine evaluation.

Matrigel, a mouse sarcoma-derived basement membrane protein mixture, is frequently used to facilitate human tumor xenograft growth in rodents. Despite its known effects on tumor growth and metastasis, its impact on tumor pathophysiology and preclinical evaluation of nanomedicines in tumor xenografts has not been reported previously. Herein bilateral MDA435 tumors were established orthotopically with (Mat+) or without (Mat-) co-injection of Matrigel. Tumor perfusion, morphology and nanoparticle retention were evaluated. As compared to Mat- tumors, Mat+tumors exhibited enhanced vascular perfusion and lymphatic flow, greater blood vessel and lymphatic growth within the tumor core, and more deformation and collapse of lymphatics in tumor-associated lymph nodes. These changes were accompanied by reduced nanoparticle retention in Mat+tumors. The results suggest that Matrigel is not a passive medium for tumor growth, but rather significantly alters long-term tumor architecture. These findings have significant implications for the evaluation of therapeutic nanomedicine in xenograft mouse models. FROM THE CLINICAL EDITOR: Matrigel is utilized in facilitating human tumor xenograft growth in rodents. The authors demonstrate that Matrigel is not a passive medium for tumor growth; instead it significantly alters long-term tumor architecture, with major implications in the evaluation of therapeutic nanomedicine in xenograft mouse models.

Remodeling Tumor Immune Microenvironment by Using Polymer-Lipid-Manganese Dioxide Nanoparticles with Radiation Therapy to Boost Immune Response of Castration-Resistant Prostate Cancer.

Despite substantial progress in the treatment of castration-resistant prostate cancer (CRPC), including radiation therapy and immunotherapy alone or in combination, the response to treatment remains poor due to the hypoxic and immunosuppressive nature of the tumor microenvironment. Herein, we exploited the bioreactivity of novel polymer-lipid manganese dioxide nanoparticles (PLMDs) to remodel the tumor immune microenvironment (TIME) by increasing the local oxygen levels and extracellular pH and enhancing radiation-induced immunogenic cell death. This study demonstrated that PLMD treatment sensitized hypoxic human and murine CRPC cells to radiation, significantly increasing radiation-induced DNA double-strand breaks and ultimately cell death, which enhanced the secretion of damage-associated molecular patterns, attributable to the induction of autophagy and endoplasmic reticulum stress. Reoxygenation via PLMDs also polarized hypoxic murine RAW264.7 macrophages toward the M1 phenotype, enhancing tumor necrosis factor alpha release, and thus reducing the viability of murine CRPC TRAMP-C2 cells. In a syngeneic TRAMP-C2 tumor model, intravenous injection of PLMDs suppressed, while radiation alone enhanced recruitment of regulatory T cells and myeloid-derived suppressor cells. Pretreatment with PLMDs followed by radiation down-regulated programmed death-ligand 1 and promoted the infiltration of antitumor CD8+ T cells and M1 macrophages to tumor sites. Taken together, TIME modulation by PLMDs plus radiation profoundly delayed tumor growth and prolonged median survival compared with radiation alone. These results suggest that PLMDs plus radiation is a promising treatment modality for improving therapeutic efficacy in radioresistant and immunosuppressive solid tumors.

Brain-Penetrating and Disease Site-Targeting Manganese Dioxide-Polymer-Lipid Hybrid Nanoparticles Remodel Microenvironment of Alzheimer's Disease by Regulating Multiple Pathological Pathways.

Finding effective disease-modifying treatment for Alzheimer's disease remains challenging due to an array of factors contributing to the loss of neural function. The current study demonstrates a new strategy, using multitargeted bioactive nanoparticles to modify the brain microenvironment to achieve therapeutic benefits in a well-characterized mouse model of Alzheimer's disease. The application of brain-penetrating manganese dioxide nanoparticles significantly reduces hypoxia, neuroinflammation, and oxidative stress; ultimately reducing levels of amyloid ß plaques within the neocortex. Analyses of molecular biomarkers and magnetic resonance imaging-based functional studies indicate that these effects improve microvessel integrity, cerebral blood flow, and cerebral lymphatic clearance of amyloid ß. These changes collectively shift the brain microenvironment toward conditions more favorable to continued neural function as demonstrated by improved cognitive function following treatment. Such multimodal disease-modifying treatment may bridge critical gaps in the therapeutic treatment of neurodegenerative disease.

Exploring the transformability of polymer-lipid hybrid nanoparticles and nanomaterial-biology interplay to facilitate tumor penetration, cellular uptake and intracellular targeting of anticancer drugs.

BACKGROUND: Successful delivery of anticancer drugs to intracellular targets requires different properties of the nanocarrier to overcome multiple transport barriers. However, few nanocarrier systems, to date, possess such properties, despite knowledge about the biological fate of inorganic and polymeric nanocarriers in relation to their fixed size, shape and surface properties. Herein, a polymer-lipid hybrid nanoparticle (PLN) system is described with size and shape transformability and its mechanisms of cellular uptake and intracellular trafficking are studied. METHODS: Pharmaceutical lipids were screened for use in transformable PLN. Mechanisms of cellular uptake and the role of fatty acid-binding proteins in intracellular trafficking of PLN were investigated in breast cancer cells. Intra-tumoral penetration and retention of doxorubicin (DOX) were evaluated by confocal microscopy. RESULTS: The lead PLNs showed time-dependent size reduction and shape change from spherical to spiky shape. This transformability of PLNs and lipid trafficking pathways facilitated intracellular transport of DOX-loaded PLN (DOX-PLN) into mitochondria and nuclei. DOX-PLN significantly increased DOX penetration and retention over free DOX or non-transformable liposomal DOX particles at 4 h post-intravenous administration. CONCLUSION: Transformability of PLN and lipid-biology interplay can be exploited to design new nanocarriers for effective drug delivery to tumor cells and intracellular targets.

A novel doxorubicin-mitomycin C co-encapsulated nanoparticle formulation exhibits anti-cancer synergy in multidrug resistant human breast cancer cells.

Anthracycline-containing treatment regimens are currently the most widely employed regimens for the management of breast cancer. These drug combinations are often designed based on non-cross resistance and minimal overlapping toxicity rather than drug synergism. Moreover, aggressive doses are normally used in chemotherapy to achieve a greater therapeutic benefit at the cost of more acute and long-term toxic effects. To increase chemotherapeutic efficacy while decreasing toxic effects, rational design of drug synergy-based regimens is needed. Our previous work showed a synergistic effect of doxorubicin (DOX) and mitomycin C (MMC) on murine breast cancer cells in vitro and improved efficacy and reduced systemic toxicity of DOX-loaded solid polymer-lipid hybrid nanoparticles (PLN) in animal models of breast cancer. Herein we have demonstrated true anticancer synergy of concurrently applied DOX and MMC, and have rationally designed PLN to effectively deliver this combination to multidrug resistant (MDR) MDA435/LCC6 human breast cancer cells. DOX-MMC co-loaded PLN were effective in killing MDR cells at 20-30-fold lower doses than the free drugs. This synergistic cell killing was correlated with enhanced induction of DNA double strand breaks that preceded apoptosis. Importantly, co-encapsulation of dual agents into a nanoparticle formulation was much more effective than concurrent application of single agent-containing PLN, demonstrating the requirement of simultaneous uptake of both drugs by the same cells to enhance the drug synergy. The rationally designed combination chemotherapeutic PLN can overcome multidrug resistance at a significantly lower dose than free drugs, exhibiting the potential to enhance chemotherapy and reduce the therapeutic limitations imposed by systemic toxicity.

Multitargeted Nanoparticles Deliver Synergistic Drugs across the Blood-Brain Barrier to Brain Metastases of Triple Negative Breast Cancer Cells and Tumor-Associated Macrophages.

Patients with brain metastases of triple negative breast cancer (TNBC) have a poor prognosis owing to the lack of targeted therapies, the aggressive nature of TNBC, and the presence of the blood-brain barrier (BBB) that blocks penetration of most drugs. Additionally, infiltration of tumor-associated macrophages (TAMs) promotes tumor progression. Here, a terpolymer-lipid hybrid nanoparticle (TPLN) system is designed with multiple targeting moieties to first undergo synchronized BBB crossing and then actively target TNBC cells and TAMs in microlesions of brain metastases. In vitro and in vivo studies demonstrate that covalently bound polysorbate 80 in the terpolymer enables the low-density lipoprotein receptor-mediated BBB crossing and TAM-targetability of the TPLN. Conjugation of cyclic internalizing peptide (iRGD) enhances cellular uptake, cytotoxicity, and drug delivery to brain metastases of integrin-overexpressing TNBC cells. iRGD-TPLN with coloaded doxorubicin (DOX) and mitomycin C (MMC) (iRGD-DMTPLN) exhibits higher efficacy in reducing metastatic burden and TAMs than nontargeted DMTPLN or a free DOX/MMC combination. iRGD-DMTPLN treatment reduces metastatic burden by 6-fold and 19-fold and increases host median survival by 1.3-fold and 1.6-fold compared to DMTPLN or free DOX/MMC treatments, respectively. These findings suggest that iRGD-DMTPLN is a promising multitargeted drug delivery system for the treatment of integrin-overexpressing brain metastases of TNBC.

Combining Tumor Microenvironment Modulating Nanoparticles with Doxorubicin to Enhance Chemotherapeutic Efficacy and Boost Antitumor Immunity.

BACKGROUND: Tumor microenvironment (TME) and associated multiple factors are found to contribute to the failures in cancer therapies, including chemo- and immunotherapy. Here we report a new multimodal strategy that uses a bioreactive multifunctional hybrid polymer-lipid encapsulated manganese dioxide nanoparticle (PLMD NP) system to remodel the TME, suppress drug resistance factors, reverse immunosuppressive conditions, and enhance chemotherapy efficacy. METHODS: The influence of PLMD NPs on enhancing cellular uptake in EMT6 mouse breast cancer cells and tumor penetration of doxorubicin (DOX) in EMT6 orthotopic breast tumor mouse model was evaluated using confocal microscopy (n = 3-4). Immunohistochemistry was employed to examine the effect of PLMD NPs on downregulating hypoxia-induced drug resistance proteins and anticancer activity of DOX (n = 3-4). The efficacy of the combination therapy with PLMD NPS and DOX was assessed in murine EMT6 (n = 15-23) and 4T1 (n = 7) orthotopic breast tumor mouse models. Rechallenge and splenocyte transfer were performed to validate the stimulation of adaptive tumor immunity in the surviving mice. RESULTS: PLMD NPs enhanced intratumoral penetration and efficacy of DOX, and reduced intratumoral expression of P-glycoprotein, p53, and carbonic anhydrase IX by 74.5%, 38.0%, and 58.8% vs saline control, respectively. Combination treatment with PLMD NPs and DOX increased the number of tumor-infiltrated CD8+ T cells and resulted in up to 60.0% complete tumor regression. Of naïve mice (n = 7) that received splenocytes from the PLMD+DOX-treated surviving mice, 57.1% completely suppressed tumor growth whereas 100% of mice that received splenocytes from DOX-treated mice (n = 3) and the control group (n = 7) showed rapid tumor growth. CONCLUSIONS: The clinically suitable PLMD NPs can effectively downregulate TME-associated drug resistance and immunosuppression. The combination therapy with PLMD NPs and DOX is a multimodal and translational treatment approach for enhancing chemotherapeutic efficacy and boosting antitumor immunity.

Chemotherapy with anticancer drugs encapsulated in solid lipid nanoparticles.

The prospect of improved cancer chemotherapy using solid lipid nanoparticles (SLN) as a drug delivery system is promising. Several obstacles frequently encountered with anticancer compounds, such as normal tissue toxicity, poor specificity and stability and a high incidence of drug-resistant tumor cells, are at least partially overcome by delivering them using SLN. The emergence of the newer forms of SLN such as polymer-lipid hybrid nanoparticles, nanostructured lipid carriers and long-circulating SLN may further expand the role of this versatile drug carrier in cancer treatment. This review focuses on the current use of SLN for the encapsulation and delivery of cytotoxic anticancer compounds. It also discusses more recent trends in the use of SLN as vehicles for delivery of chemosensitizers and cytotoxic therapeutic molecules. It is anticipated that, in the near future, SLN will be further improved to deliver anticancer compounds in a more efficient, specific and safer manner.

On the synergistic effect of doxorubicin and mitomycin C against breast cancer cells.

The combination of doxorubicin and mitomycin C has been shown previously to result in supra-additive tumor cell killing in vitro in both murine and human breast cancer cells and in vivo against murine breast cancer cells. Median effect analysis was used to determine the significance and degree of interaction. The origin of this synergy was sought by evaluating the contribution of membrane efflux pump modulation, formaldehyde production, reactive oxygen species, DNA cross-linking, and DNA double-strand breaks to this effect. The interaction of mitomycin C and doxorubicin in vitro was found to be a true synergy whose mechanism was efflux pump-independent. DNA cross-links were only found to increase additively with co-administration of the drugs; however, a supra-additive increase in DNA double-strand breaks was observed. The results suggest that poisoning of topoisomerase IIalpha by doxorubicin may interact with drug-induced DNA cross-links to enhance the formation of DNA double-strand breaks. This interaction, together with glutathione depletion and mitomycin C-derived formaldehyde, may be the underlying mechanism(s) of the synergy observed between mitomycin C and doxorubicin.

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice.

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

In vitro toxicity to breast cancer cells of microsphere-delivered mitomycin C and its combination with doxorubicin.

To better understand and design microsphere systems for the locoregional delivery of anticancer drug combinations to solid tumors, (1) the cytotoxicity of microsphere-delivered mitomycin C (MMC) was evaluated and (2) various schedules of MMC and doxorubicin (Dox) were tested for their toxicity in vitro towards a murine breast cancer cell-line, EMT6. To accomplish the former MMC was loaded onto oxidized sulfopropyl dextran microspheres, released in a pH 7.4 buffer solution and tested for its potency against EMT6 cells versus a standard MMC solution. For the latter EMT6 cells were exposed to MMC or Dox as single agents or together using various drug concentrations and schedules. The efficacy of the treatments was measured using a clonogenic assay. MMC released from the microspheres showed similar activity against EMT6 cells to freshly prepared MMC solutions. Greater-than-additive toxicity was observed when MMC was given either simultaneously or after Dox exposure. In contrast, administration of MMC before Dox exposure resulted in toxicity that ranged from additive to sub-additive; this reduced toxicity was mainly due to increasing cell density arising from the design of the assay. These results help explain our previous in vivo investigations using microsphere-delivered combinations of the same agents in EMT6 solid tumors.

Hybrid Manganese Dioxide Nanoparticles Potentiate Radiation Therapy by Modulating Tumor Hypoxia.

Hypoxia in the tumor microenvironment (TME) mediates resistance to radiotherapy and contributes to poor prognosis in patients receiving radiotherapy. Here we report the design of clinically suitable formulations of hybrid manganese dioxide (MnO2) nanoparticles (MDNP) using biocompatible materials to reoxygenate the TME by reacting with endogenous H2O2 MDNP containing hydrophilic terpolymer-protein-MnO2 or hydrophobic polymer-lipid-MnO2 provided different oxygen generation rates in the TME relevant to different clinical settings. In highly hypoxic murine or human xenograft breast tumor models, we found that administering either MDNP formulation before radiotherapy modulated tumor hypoxia and increased radiotherapy efficacy, acting to reduce tumor growth, VEGF expression, and vascular density. MDNP treatment also increased apoptosis and DNA double strand breaks, increasing median host survival 3- to 5-fold. Notably, in the murine model, approximately 40% of tumor-bearing mice were tumor-free after a single treatment with MDNPs plus radiotherapy at a 2.5-fold lower dose than required to achieve the same curative treatment without MDNPs. Overall, our findings offer a preclinical proof of concept for the use of MDNP formulations as effective radiotherapy adjuvants. Cancer Res; 76(22); 6643-56. ©2016 AACR.

Biodegradable dextran-based microspheres for delivery of anticancer drug mitomycin C.

The purpose of this work was to develop a biodegradable microsphere (MS) system for delivering mitomycin C (MMC). Various dextran-based MS systems were investigated for their loading and release characteristics, including nonionic MS, sulfopropyl dextran microspheres (SP-MS) with low or high cross-linking density, oxidized SP-MS (Ox-MS), and hydrophobically modified SP-MS. SP-MS were chemically modified by oxidation with sodium periodate or by reaction with anhydride. The chemical structure of modified SP-MS and MMC-loaded MS (MMC-MS) were examined using Fourier transform infrared (FTIR) and solid-state nuclear magnetic resonance (NMR) spectrophotometry. Drug release was conducted at 37 degrees C in aqueous solutions of 0.15 m phosphate buffer solution. The kinetics of drug absorption and release and the stability of MMC after loading and release were determined by spectrophotometry and high-performance liquid chromatography. Ionic SP-MS exhibited a higher drug-loading rate and capacity when compared to nonionic MS, while hydrophobically modified SP-MS showed an even greater loading capacity than SP-MS. These results suggest that both ionic complexation and hydrophobic interaction were important factors in MMC loading. The Ox-MS system demonstrated higher drug-loading capacity, more fractional drug release and a longer time to reach release equilibrium as compared to other investigated MS systems. Under optimized reaction and loading conditions, MMC released from Ox-MS was found to be unaltered. This work demonstrates that the Ox-MS system is a potentially useful system for the delivery of MMC.