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Radiotherapy (RT) is one of major therapeutic modalities in combating breast cancer. In RT, ionizing radiation is employed to induce DNA double-strand breaks (DSBs) as a primary mechanism that causes cancer cell death. However, the induced DNA damage can also trigger the activation of DNA repair mechanisms, reducing the efficacy of RT treatment. Given the pivotal role of RAD50 protein in the radiation-responsive DNA repair pathways involving DSBs, we developed a novel polymer-lipid based nanoparticle formulation containing RAD50-silencing RNA (RAD50-siRNA-NPs) and evaluated its effect on the RAD50 downregulation as well as cellular and tumoral responses to ionizing radiation using human triple-negative breast cancer as a model. The RAD50-siRNA-NPs successfully preserved the activity of the siRNA, facilitated its internalization by cancer cells via endocytosis, and enabled its lysosomal escape. The nanoparticles significantly reduced RAD50 expression, whereas RT alone strongly increased RAD50 levels at 24 h. Pretreatment with RAD50-siRNA-NPs sensitized the cancer cells to RT with ∼2-fold higher level of initial DNA DSBs as determined by a γH2AX biomarker and a 2.5-fold lower radiation dose to achieve 50 % colony reduction. Intratumoral administration of RAD50-siRNA-NPs led to a remarkable 53 % knockdown in RAD50. The pretreatment with RAD50-siRNA-NPs followed by RT resulted in approximately a 2-fold increase in DNA DSBs, a 4.5-fold increase in cancer cell apoptosis, and 2.5-fold increase in tumor growth inhibition compared to RT alone. The results of this work demonstrate that RAD50 silencing by RAD50-siRNA-NPs can disrupt RT-induced DNA damage repair mechanisms, thereby significantly enhancing the radiation sensitivity of TNBC MDA-MB-231 cells in vitro and in orthotopic tumors as measured by colony forming and tumor regrowth assays, respectively., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

A novel brain-targeted and reactive oxygen species-activatable manganese dioxide containing nanoparticle system functionalized with anti-amyloid-β antibody (named aAβ-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 aAβ-BTRA-NC in a transgenic TgCRND8 mouse AD model, wild type (WT) littermate, and CD-1 mice. Dose-ascending studies demonstrated that aAβ-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 aAβ-BTRA-NC did not cause any toxicity in major organs. Immunotoxicity data showed that aAβ-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 aAβ-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 aAβ-BTRA-NC followed a 2-compartmental model with faster clearance in the AD mice than in the WT mice. The results suggest that aAβ-BTRA-NC exhibits a strong safety profile as a nanotheranostic agent which warrants more robust preclinical development for future clinical applications.

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 Mn 2+ ions, enhancing T 1 -weight MRI contrast via the reaction with H 2 O 2 rich in the acidic tumor microenvironment. Compared to the clinically used GBCA, Gadovist®1.0, TPL-MDNP generated stronger T 1 -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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

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., (Copyright © 2023 Abdulmottaleb E. Zetrini et al.)

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 β 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 β. 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., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

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.

Introduction: Radiation therapy (RT) is a major modality for the treatment of prostate cancer (PCa), especially castration-resistant PCa (CRPC). However, hypoxia, often seen in PCa tumors, leads to radiation-resistance. This work investigates the effect of a novel oxygen-generating polymer-lipid manganese dioxide nanoparticle (PLMDs) on improving RT outcomes in CRPC xenograft models by modulating the tumor microenvironment (TME) both before and after RT., Materials and Methods: Human PC3 and DU145 PCa cells were used to investigate clonogenic inhibition and DNA repair pathways in vitro. Tumor hypoxia and post-RT angiogenesis were evaluated in a PC3-bearing SCID mouse model. PC3 and DU145 xenografts were used to study the efficacy of PLMD in combination with single or fractionated RT., Results: PLMD plus RT significantly inhibited clonogenic potential, increased DNA double-strand breaks, and reduced DNA damage repair in hypoxic PC3 and DU145 cells as compared to RT alone. PLMD significantly reduced hypoxia-positive areas, hypoxia induced factor 1α (HIF-1α) expression, and protein carbonyl levels (a measure of oxidative stress). Application of PLMD with RT decreased RT-induced angiogenic biomarkers by up to 3-fold. Treatment of the human CRPC xenografts with PLMD plus RT (single or fractionated doses) significantly prolonged median survival of the host compared to RT alone resulting in up to a 40% curative rate., Conclusion: PLMD treatment modulated TME and sensitized hypoxic human CRPC cells to RT thus enhancing the efficacy of RT. These results confirmed the potential of PLMD as an adjuvant to RT for the treatment of hypoxic CRPC., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 2 3 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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

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.

Lung metastasis is the major cause of death in patients with triple negative breast cancer (TNBC), an aggressive subtype of breast cancer with no effective therapy at present. It has been proposed that dual-targeted therapy, ie, targeting chemotherapeutic agents to both tumor vasculature and cancer cells, may offer some advantages. The present work was aimed to develop a dual-targeted synergistic drug combination nanomedicine for the treatment of lung metastases of TNBC. Thus, Arg-Gly-Asp peptide (RGD)-conjugated, doxorubicin (DOX) and mitomycin C (MMC) co-loaded polymer-lipid hybrid nanoparticles (RGD-DMPLN) were prepared and characterized. The synergism between DOX and MMC and the effect of RGD-DMPLN on cell morphology and cell viability were evaluated in human MDA-MB-231 cells in vitro. The optimal RGD density on nanoparticles (NPs) was identified based on the biodistribution and tumor accumulation of the NPs in a murine lung metastatic model of MDA-MB-231 cells. The microscopic distribution of RGD-conjugated NPs in lung metastases was examined using confocal microscopy. The anticancer efficacy of RGD-DMPLN was investigated in the lung metastatic model. A synergistic ratio of DOX and MMC was found in the MDA-MB-231 human TNBC cells. RGD-DMPLN induced morphological changes and enhanced cytotoxicity in vitro. NPs with a median RGD density showed the highest accumulation in lung metastases by targeting both tumor vasculature and cancer cells. Compared to free drugs, RGD-DMPLN exhibited significantly low toxicity to the host, liver and heart. Compared to non-targeted DMPLN or free drugs, administration of RGD-DMPLN (10 mg/kg, iv) resulted in a 4.7-fold and 31-fold reduction in the burden of lung metastases measured by bioluminescence imaging, a 2.4-fold and 4.0-fold reduction in the lung metastasis area index, and a 35% and 57% longer median survival time, respectively. Dual-targeted RGD-DMPLN, with optimal RGD density, significantly inhibited the progression of lung metastasis and extended host survival.

Brain metastasis is a fatal disease with limited treatment options and very short survival. Although systemic chemotherapy has some effect on peripheral metastases of breast cancer, it is ineffective in treating brain metastasis due largely to the blood-brain barrier (BBB). Here we developed a BBB-penetrating amphiphilic polymer-lipid nanoparticle (NP) system that efficiently delivered anti-mitotic drug docetaxel (DTX) for the treatment of brain metastasis of triple negative breast cancer (TNBC). We evaluated the biodistribution, brain accumulation, pharmacokinetics and efficacy of DTX-NP in a mouse model of brain metastasis of TNBC. Confocal fluorescence microscopy revealed extravasation of dye-loaded NPs from intact brain microvessels in healthy mice. DTX-NP also extravasated from brain microvessels and accumulated in micrometastasis lesions in the brain. Intravenously injected DTX-NPs increased the blood circulation time of DTX by 5.5-fold and the AUC 0-24h in tumor-bearing brain by 5-fold compared to the clinically used DTX formulation Taxotere® . The kinetics of NPs in the brain, determined by ex vivo fluorescence imaging, showed synchronization with DTX kinetics in the brain measured by LC-MS/MS. This result confirmed successful delivery of DTX by the NPs into the brain and suggested that ex vivo fluorescence imaging of NP could be an effective and quick means for probing drug disposition in the brain. Treatment with the DTX-NP formulation delayed tumor growth by 11-fold and prolonged median survival of tumor-bearing mice by 94% compared to an equivalent dose of Taxotere®, without inducing histological changes in the major organs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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 (MnO 2 ) nanoparticles (MDNP) using biocompatible materials to reoxygenate the TME by reacting with endogenous H 2 O 2 MDNP containing hydrophilic terpolymer-protein-MnO 2 or hydrophobic polymer-lipid-MnO 2 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., (©2016 American Association for Cancer Research.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study was performed to optimize the formulation of polymer-lipid hybrid nanoparticles (PLN) for the delivery of an ionic water-soluble drug, verapamil hydrochloride (VRP) and to investigate the roles of formulation factors. Modeling and optimization were conducted based on a spherical central composite design. Three formulation factors, i.e., weight ratio of drug to lipid (X1), and concentrations of Tween 80 (X2) and Pluronic F68 (X3), were chosen as independent variables. Drug loading efficiency (Y1) and mean particle size (Y2) of PLN were selected as dependent variables. The predictive performance of artificial neural networks (ANN) and the response surface methodology (RSM) were compared. As ANN was found to exhibit better recognition and generalization capability over RSM, multi-objective optimization of PLN was then conducted based upon the validated ANN models and continuous genetic algorithms (GA). The optimal PLN possess a high drug loading efficiency (92.4%, w/w) and a small mean particle size (∼100nm). The predicted response variables matched well with the observed results. The three formulation factors exhibited different effects on the properties of PLN. ANN in coordination with continuous GA represent an effective and efficient approach to optimize the PLN formulation of VRP with desired properties., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Multifunctional nanoparticles (NPs) have found important applications in diagnosis, chemotherapy, and image-guided surgery of tumors. In this work, we have developed polymeric theranostic NPs (PTNPs) containing the anticancer drug docetaxel (DTX), a fluorescent dye, and magnetic manganese oxide (MnO) NPs for dual modal imaging and chemotherapy. PTNPs ~150 nm in diameter were synthesized by co-loading hydrophobic DTX and MnO NPs ~5 nm in diameter, into the matrix of a fluorescent dye-labeled amphiphilic polymer. The PTNPs enabled high loading efficiency and sustained in vitro release of DTX. Energy-dependent cellular uptake and extended cytoplasmic retention of the PTNPs in MDA-MB-231 human breast cancer cells were observed by fluorescence microscopy examination. DTX-loaded PTNPs exhibited higher cytotoxicity than free DTX with a 3 to 4.4-fold decrease in drug dose required for 50% cell growth inhibition. The hydrophilic backbone of the amphiphilic polymer improved the fluidity of PTNPs which enhanced the longitudinal relaxivity (r1) of loaded MnO NPs by 2.7-fold with r1=2.4mM(-1)s(-1). Whole body fluorescence imaging (FI) and magnetic resonance imaging (MRI) showed significant accumulation and prolonged retention of PTNPs in orthotopic MDA-MB-231 breast tumors. These results suggest that the new amphiphilic polymer-based PTNP system, able to simultaneously deliver a poorly soluble anticancer drug, enhance MRI contrast, and stain tumor tissue by fluorescence, is a good candidate for cancer theranostic applications., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Glucose oxidase (GOX) encapsulated in alginate-chitosan microspheres (GOX-MS) was shown in our previous work to produce reactive oxygen species (ROS) in situ and exhibit anticancer effects in vitro and in vivo. The purpose of present work was to optimize the design and thus enhance the efficacy of GOX-MS against multidrug resistant (MDR) cancer cells. GOX-MS with different mean diameters of 4, 20 or 140 μm were prepared using an emulsification-internal gelation-adsorption-chitosan coating method with varying compositions and conditions. The GOX loading efficiency, loading level, relative bioactivity of GOX-MS, and GOX leakage were determined and optimal chitosan concentrations in the coating solution were identified. The influence of particle size on cellular uptake, ROS generation, cytotoxicity and their underlying mechanisms was investigated. At the same GOX dose and incubation time, smaller sized GOX-MS produced larger amounts of H2O2 in cell culture medium and greater cytotoxicity toward murine breast cancer MDR (EMT6/AR1.0) and wild type (EMT6/WT) cells. Fluorescence and confocal laser scanning microscopy revealed significant uptake of small sized (4 μm) GOX-MS by both MDR and WT cells, but no cellular uptake of large (140 μm) GOX-MS. The GOX-MS were equally effective in killing both MDR cells and WT cells. The cytotoxicity of the GOX formulations was positively correlated with membrane damage and lipid peroxidation. GOX-MS induced greater membrane damage and lipid peroxidation in MDR cells than the WT cells. These results suggest that the optimized, small micron-sized GOX-MS are highly effective against MDR breast cancer cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

αvβ3 integrin receptors expressed on cancer cell surfaces play a crucial role in promoting tumor angiogenesis and cancer cell metastasis. Thus, cyclic arginyl-glycyl-aspartic acid (cRGD) peptides have been explored as a αvβ3 integrin receptor-specific targeting moiety for the targeted delivery of nanoparticle-loaded therapeutics. However, our previous study showed that cyclic RGD could act as a double-edged sword that, on one hand, extended the retention of cRGD-modified solid lipid nanoparticles (RGD-SLNs) at αvβ3 integrin receptor overexpressing breast carcinoma, and yet on the other hand, decreased the amount of tumor accumulation of RGD-SLNs attributable to the greater uptake by the mononuclear phagocyte system (MPS). Therefore, we aimed to optimize the RGD-decorated nanoparticle systems for (1) inhibiting αvβ3 integrin receptor overexpressing tumor cell metastasis and (2) increasing nanoparticle accumulation to tumor site. SLNs with cRGD content ranging from 0 to 10 % mol of total polyethyleneglycol (PEG) chains were synthesized. The binding of RGD-SLNs with αvβ3 integrin receptors increased with increasing cRGD concentration on the nanoparticles. RGD-SLNs were demonstrated to inhibit MDA-MB-231 cell adhesion to fibronectin and invasion through Matrigel. In vivo whole-body fluorescence imaging revealed that 1 % cRGD on the SLNs' surface had maximum tumor accumulation with extended tumor retention among all formulations tested in an orthotopic MDA-MB-231/EGFP breast tumor model. This work has laid a foundation for further development of anticancer drug-loaded optimized cRGD nanoparticle formulations for the treatment of breast cancer metastasis.

Metastatic brain cancers, in particular cancers with multiple lesions, are one of the most difficult malignancies to treat owing to their location and aggressiveness. Chemotherapy for brain metastases offers some hope. However, its efficacy is severely limited as most chemotherapeutic agents are incapable of crossing the blood-brain barrier (BBB) efficiently. Thus, a multifunctional nanotheranostic system based on poly(methacrylic acid)-polysorbate 80-grafted-starch was designed herein for the delivery of BBB-impermeable imaging and therapeutic agents to brain metastases of breast cancer. In vivo magnetic resonance imaging and confocal fluorescence microscopy were used to confirm extravasation of gadolinium and dye-loaded nanoparticles from intact brain microvessels in healthy mice. The targetability of doxorubicin (Dox)-loaded nanoparticles to intracranially established brain metastases of breast cancer was evaluated using whole body and ex vivo fluorescence imaging of the brain. Coexistence of nanoparticles and Dox in brain metastatic lesions was further confirmed by histological and microscopic examination of dissected brain tissue. Immuno-histochemical staining for caspase-3 and terminal-deoxynucleotidyl transferase dUTP nick end labeling for DNA fragmentation in tumor-bearing brain sections revealed that Dox-loaded nanoparticles selectively induced cancer cell apoptosis 24 h post-injection, while sparing normal brain cells from harm. Such effects were not observed in the mice treated with free Dox. Treatment with Dox-loaded nanoparticles significantly inhibited brain tumor growth compared to free Dox at the same dose as assessed by in vivo bioluminescence imaging of the brain metastases. These findings suggest that the multifunctional nanoparticles are promising for the treatment of brain metastases.

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.

Insufficient oxygenation (hypoxia), acidic pH (acidosis), and elevated levels of reactive oxygen species (ROS), such as H2O2, are characteristic abnormalities of the tumor microenvironment (TME). These abnormalities promote tumor aggressiveness, metastasis, and resistance to therapies. To date, there is no treatment available for comprehensive modulation of the TME. Approaches so far have been limited to regulating hypoxia, acidosis, or ROS individually, without accounting for their interdependent effects on tumor progression and response to treatments. Hence we have engineered multifunctional and colloidally stable bioinorganic nanoparticles composed of polyelectrolyte-albumin complex and MnO2 nanoparticles (A-MnO2 NPs) and utilized the reactivity of MnO2 toward peroxides for regulation of the TME with simultaneous oxygen generation and pH increase. In vitro studies showed that these NPs can generate oxygen by reacting with H2O2 produced by cancer cells under hypoxic conditions. A-MnO2 NPs simultaneously increased tumor oxygenation by 45% while increasing tumor pH from pH 6.7 to pH 7.2 by reacting with endogenous H2O2 produced within the tumor in a murine breast tumor model. Intratumoral treatment with NPs also led to the downregulation of two major regulators in tumor progression and aggressiveness, that is, hypoxia-inducible factor-1 alpha and vascular endothelial growth factor in the tumor. Combination treatment of the tumors with NPs and ionizing radiation significantly inhibited breast tumor growth, increased DNA double strand breaks and cancer cell death as compared to radiation therapy alone. These results suggest great potential of A-MnO2 NPs for modulation of the TME and enhancement of radiation response in the treatment of cancer.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Multidrug resistance (MDR) and drug toxicity are two major factors responsible for the failure of cancer chemotherapy. Herein the efficacy and safety of combination therapy using doxorubicin (Dox, D)-mitomycin C (MMC, M) co-loaded stealth polymer-lipid hybrid nanoparticles (DMsPLNs) were evaluated in sensitive and MDR human mammary tumor xenografts. DMsPLN demonstrated enhanced efficacy compared to liposomal Dox (PLD) with up to a 3-fold increase in animal life span, a 10-20% tumor cure rate, undetectable normal tissue toxicity and decreased tumor angiogenesis. These results suggest DMsPLN have potential as an effective treatment of breast cancer., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

A new multifunctional terpolymeric system for simultaneous imaging and drug delivery has been recently developed in our laboratory. Herein we report the investigation of terpolymeric contrast agent for magnetic resonance imaging and doxorubicin (Dox) delivery. The polymer was synthesized by graft polymerization of methacrylic acid (MAA) and polysorbate 80 (PS 80) onto starch with multiple, chemically bound diethylenetriaminepenta acetic acid (DTPA) groups for gadolinium chelating. The terpolymer self-assembled to form nanoparticles upon addition of doxorubicin which binds with the PMAA chain. The physicochemical, biological and pharmacokinetic properties of the polymeric system were characterized and their contrast enhancement capability was evaluated in vitro and in vivo. The polymer was able to load gadolinium with high thermodynamic stability and exhibited low cytotoxicity. The Gd-loaded polymer (PolyGd), and Gd-Dox co-loaded nanoparticles (PolyGd-Dox) significantly enhanced MR signals, with ionic T1 relaxivities 3-5 times higher than those from Omniscan®, a small molecule contrast agent. In vivo studies showed superior and prolonged contrast enhancement compared to Omniscan® at one fourth the equivalent dose, without adverse effects. The PolyGd and PolyGd-Dox accumulated in the tumor and painted the tumor boundaries clearly for at least 48h. The PolyGd also enhanced angiogram contrast with contrast to noise ratio values of up to 55-fold and a blood half-life time of 200min. Seven days after intravenous administration, only relatively small amounts of gadolinium could be detected in the major organs of the mice (supplementary materials). These results suggest that the new terpolymeric system is useful as a theranostic platform for contrast enhanced MR imaging of vasculature and tumor as well as Dox delivery., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Successful development of a nanoparticulate system for cancer chemotherapy requires detailed knowledge of its biodistribution, clearance and anti-tumour efficacy in vivo. Herein we developed new bi-functional nanoparticles for simultaneous in vivo optical imaging and delivery of the anticancer drug doxorubicin (Dox) for enhanced chemotherapy. Two types of nanoparticles were synthesized, namely preformed nanoparticles (PF-NPs) and self-assembled nanoparticles (SA-NPs). The PF-NPs were prepared by cross-linking graft polymerization of methacrylic acid and polysorbate 80 with starch (PMAA-PS 80-g-St) and then loading the particles with Dox. The SA-NPs were formed upon addition of Dox to non-cross-linked PMAA-PS 80-g-St. A near infrared fluorescent probe was conjugated with the PMAA unit of the nanoparticles. The biodistribution, tumour targeting and pharmacokinetics of the Dox-loaded nanoparticles in mice were determined by in vivo/ex vivo fluorescence imaging and ex vivo fluorescence microscopy. The anti-tumour efficacy of the nanoparticles was investigated using a murine orthotopic breast cancer model. PF-NPs had an average hydrodynamic diameter and zeta potential of 137 ± 3 nm and -38 ± 1 mV, respectively. These values were measured at 62 ± 5 nm and -35 ± 5 mV for SA-NPs. PF-NPs exhibited a porous morphology while the SA-NPs appeared to have a denser structure. SA-NPs outperformed the PF-NPs in terms of blood circulation, tumour uptake and penetration. PF-NPs and SA-NPs exhibited no systemic toxicity and inhibited tumour growth significantly better than the free Dox solution with SA-NPs being the best, attributable to their excellent tumour uptake and penetration. This work demonstrates the usefulness of these bi-functional nanoparticles as nanotheranostics.

This work investigated the capability of a new nanoparticulate system, based on terpolymer of starch, polymethacrylic acid and polysorbate 80, to load and release doxorubicin (Dox) as a function of pH and to evaluate the anticancer activity of Dox-loaded nanoparticles (Dox-NPs) to overcome multidrug resistance (MDR) in human breast cancer cells in vitro. The Dox-NPs were characterized by Fourier transform infrared spectroscopy (FTIR), isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The cellular uptake and cytotoxicity of the Dox-loaded nanoparticles were investigated using fluorescence microscopy, flow cytometry, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The nanoparticles were able to load up to 49.7±0.3% of Dox with a high loading efficiency of 99.9±0.1%, while maintaining good colloidal stability. The nanoparticles released Dox at a higher rate at acidic pH attributable to weaker Dox-polymer molecular interactions evidenced by ITC. The Dox-NPs were taken up by the cancer cells in vitro and significantly enhanced the cytotoxicity of Dox against human MDR1 cells with up to a 20-fold decrease in the IC50 values. The results suggest that the new terpolymeric nanoparticles are a promising vehicle for the controlled delivery of Dox for treatment of drug resistant breast cancer., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The overexpression of α(v) β(3) integrin receptors on tumor cells and tumor vascular endothelium makes it a useful target for imaging, chemotherapy and anti-angiogenic therapy. However integrin-targeted delivery of therapeutics by nanoparticles have provided only marginal, if any, enhancement of therapeutic effect. This work was thus focused on the development of novel α(v) β(3) -targeted near infrared light-emitting solid lipid nanoparticles (SLN) through conjugation to the α(v) β(3) integrin-specific ligand cyclic Arg-Gly-Asp (cRGD), and the assessment of the effects of α(v) β(3) targeting on nanoparticle biodistribution. Since our previously developed non-targeted "stealth" SLN showed little hepatic accumulation, unlike most reported liposomes and micelles, they served as a reference for quantifying the effects of cRGD-conjugation on tumor uptake and whole animal biodistribution of SLN. Non-targeted SLN, actively targeted (RGD-SLN) and blocked RGD-SLN were prepared to contain near infrared quantum dots for live animal imaging. They were injected intravenously to nude mice bearing xenograft orthotopic human breast tumors or dorsal window chamber breast tumors. Tumor micropharmacokinetics of various SLN formulations were determined using intravital microscopy, and whole animal biodistribution was followed over time by optical imaging. The active tumor targeting with cRGD was found to be a "double-edged sword": while the specificity of RGD-SLN accumulation in tumor blood vessels and their tumor residence time increased, their distribution in the liver, spleen, and kidneys was significantly greater than the non-targeted SLN, leaving a smaller amount of nanoparticles in the tumor tissue. Nevertheless the enhanced specificity and retention of RGD-SLN in tumor neovasculature could make this novel formulation useful for tumor neovascular-specific therapies and imaging applications., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Multidrug resistance (MDR) in cancer cells can involve overexpression of different types of membrane drug efflux pumps and other drug resistance mechanisms. Hence, inhibition of one resistance mechanism may not be therapeutically effective. Previously we demonstrated a new polymer lipid hybrid nanoparticle (PLN) system was able to circumvent drug resistance of P-glycoprotein (P-gp) overexpressing breast cancer cells. The objectives of the present study were 2-fold: (1) to evaluate the ability of the PLN system to overcome two other membrane efflux pumps-multidrug resistance protein 1 (MRP1+) and breast cancer resistance protein (BCRP+) overexpressed on human breast cancer cell lines MCF7 VP (MRP1+) and MCF7 MX (BCRP+); and (2) to evaluate possible synergistic effects of doxorubicin (Dox)-mitomycin C (MMC) in these cell lines. These objectives were accomplished by measuring in vitro cellular uptake, intracellular trafficking, and cytotoxicity (using a clonogenic assay and median effect analysis), of Dox, MMC, or Dox-MMC co-loaded PLN. Treatment of MDR cells with PLN encapsulating single anticancer agents significantly enhanced cell kill compared to free Dox or MMC solutions. Dox-MMC co-loaded PLN were 20-30-folds more effective in killing MDR cells than free drugs. Co-encapsulated Dox-MMC was more effective in killing MDR cells than single agent-encapsulated PLN. Microscopic images showed perinuclear localization of fluorescently labelled PLN in all cell lines. These results are consistent with our previous results for P-gp overexpressing breast cancer cells suggesting the PLN system can overcome multiple types of membrane efflux pumps increasing the cytotoxicity of Dox-MMC at significantly lower doses than free drugs.

For effective and safe thermotherapy, real-time, accurate, three-dimensional tissue thermometry is required. Magnetic resonance imaging (MRI)-based thermometry in combination with current temperature responsive contrast agents only provides an 'off-on' signal at a certain temperature, not indicating temperature increases beyond the desired therapeutic levels. To overcome this limitation, a novel Gd-chelated hydrogel-lipid hybrid nanoparticle (HLN) formulation was developed that provides an 'off-on-off' signal defining a thermometric window for MR thermometry. Novel thermally responsive poly(N-isopropylacrylamide-co-acrylamide) (NIPAM-co-AM) hydrogel nanoparticles (<15 nm) with bisallylamidodiethylenetriaminetriacetic acid, a novel crosslinker with Gd(3+) chelation functionality, were synthesized. The Gd-hydrogel nanoparticles were encapsulated in a solid lipid nanoparticle matrix that prevented T(1)-weighted contrast signal enhancement. Melting of the matrix lipid freed the Gd-hydrogel nanoparticles into the bulk water and an 'off-on' contrast signal enhancement occurred. As the temperature was further increased to temperatures greater than, the volume phase transition temperature of the hydrogel nanoparticles, they collapsed and provided an 'on-off' signal diminution. Both the 'off-on' and the 'on-off' transition temperature could be tailored by changing the lipid matrix and altering the NIPAM/AM ratio in the hydrogel, respectively. This allowed MRI thermometry of different temperature windows using the Gd-HLN system., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Despite broad applications of quantum dots (QDs) in vitro, severe toxicity and dominant liver uptake have limited their clinical application. QDs that excite and emit in the ultraviolet and visible regions have limited in vivo applicability due to significant optical interference exerted by biological fluids and tissues. Hence we devised a new biocompatible hybrid fluorophore composed of near-infrared-emitting PbSe quantum dots encapsulated in solid fatty ester nanoparticles (QD-FEN) for in vivo imaging. The quantum yield and tissue penetration depth of the QD-FEN were characterized, and their biological fate was examined in a breast tumor-bearing animal model. It was found for the first time that chemical modification of the headgroup of QD-encapsulating organic fatty acids was a must as these groups quenched the photoluminescence of PbSe nanocrystals. The use of fatty esters enhanced aqueous quantum yields of PbSe QDs up to ∼45%, which was 50% higher than that of water-soluble PbSe nanocrystals in an aqueous medium. As a result, a greater than previously reported tissue penetration depth of fluorescence was recorded at 710 nm/840 nm excitation/emission wavelengths. The QD-FEN had much lower short-term cytotoxicity compared to nonencapsulated water-soluble QDs. More importantly, reduced liver uptake, increased tumor retention, lack of toxic response, and nearly complete clearance of QD-FEN from the tested animals was demonstrated. With a combination of near-infrared spectral properties, enhanced optical properties,and significantly improved biosafety profile, this novel hybrid nanoparticulate fluorophore system demonstrably provides real-time, deep-tissue fluorescent imaging of live animals, laying a foundation for further development toward clinical application.

Multidrug resistance (MDR) is one of the main challenges in the treatment of breast cancer. A new microsphere formulation able to generate reactive oxygen species (ROS) locally was thus investigated for circumventing MDR in breast cancer cells in this work. Glucose oxidase (GOX) was encapsulated in alginate/chitosan hydrogel microspheres (ACMS-GOX). The in vitro cytotoxicity of ACMS-GOX to murine breast cancer EMT6/AR1.0 cells, which overexpress P-glycoprotein (P-gp), was evaluated by a clonogenic assay. The mechanism of the cytotoxicity of ACMS-GOX was investigated by using various extracellular and intracellular ROS scavengers and antioxidant enzyme inhibitors. The effect of lipid peroxidation and cellular uptake of GOX was also evaluated. ACMS-GOX exhibited similar dose and time-dependent cytotoxicity to EMT6/AR1.0 cells as to their wild-type EMT6/WT parent cells, in effect circumventing the MDR phenotype of EMT6/AR1.0 cells. Extracellular H(2)O(2) and intracellular hydroxyl radical were found to play critical roles in the cytotoxicity of ACMS-GOX. Cellular uptake of GOX was negligible and thus not responsible for intracellular ROS generation. Combining ACMS-GOX with intracellular antioxidant inhibitors-enhanced cytoxicity. This work demonstrates that the ACMS-GOX are effective in circumventing P-gp-mediated MDR in breast cancer cells.