Claudia R. Gordijo

Hybrid Quantum Dot-Fatty Ester Stealth Nanoparticles: Toward Clinically Relevant in Vivo Optical Imaging of Deep Tissue

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.

Manganese oxide and docetaxel co-loaded fluorescent polymer nanoparticles for dual modal imaging and chemotherapy of breast cancer.

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.

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.

A New Bio-Inorganic Nanocomposite Membrane for Glucose-Modulated Release of Insulin

This work focuses on the development of a new bio-inorganic nanocomposite glucose-responsive membrane to be applied as a single self-regulated platform for insulin delivery. Crosslinked bovine serum albumin (BSA)-based membranes were prepared containing impregnated pH-responsive poly(N-isopropyl acrylamide-co-methacrylic acid) nanoparticles (hydrogel NPs), glucose oxidase (GOx), catalase (CAT), with or without MnO 2 NPs. The membrane acts as a glucose sensor and insulin release attenuator. In this system glucose is oxidized by GOx to produce gluconic acid, which regulates the permeability of the membrane to insulin. CAT and/or MnO 2 NPs are introduced into the membrane in order to quench unwanted H 2 O 2 produced by GOx turnover cycles, which can cause inactivation of GOx and toxicity. The glucose-modulated insulin release through the membrane is determined by alternating glucose concentration between 100 – 400 mg/dL (normal and hyperglycemic levels, respectively). The results show that the combination of CAT and MnO 2 NPs in the membrane formulation leads to better efficiency in quenching the H 2 O 2 and better long-term stability of GOx than using either alone. Very small amounts of insulin permeate though the membrane at the normal blood glucose level while a four-fold increase in the release rate is observed when glucose concentration is raised to a hyperglycemic level. The release rate of insulin drops when the glucose level is reduced to a normal value. These results demonstrate the self-regulated capability of the system.

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.

Abstract 5218: Boosting doxorubicin-induced immunogenic cell death by using tumor microenvironment modulating nanoparticles

Background: Immunogenic cell death (ICD) is a cell death modality which triggers an immune response against dead-cell antigens, particularly when they derive from cancer cells. ICD has been reported to be highly induced by anthracycline drugs, such as doxorubicin (DOX), via facilitating tumor antigen presentation to and activation of T cells. However, these immunogenic effects may be compromised due to the immunosuppressive tumor microenvironment (TME), such as excess ROS, particularly H 2 O 2, produced by tumor cells and dysfunctional macrophages in the tumor. We hypothesize that modulating immunosuppressive factors in the TME can enhance tumor response to chemotherapy and boost immunity. Specifically, polymer-lipid encapsulated manganese dioxide nanoparticles (PLMD NPs) are used to reduce tumor hypoxia and ROS by scavenging H 2 O 2 and generating oxygen. Antitumor immunity against the cancer cells can be enhanced by the combination therapy with PLMD and DOX. Methods: PLMD NPs were prepared by dispersing MnO 2 precursor particles in melted lipid and polymer in a surfactant-containing aqueous medium. The efficacy of the combination treatment was evaluated in immunocompetent mice-bearing orthotopic murine EMT6 breast tumor. The mice were treated by intravenous (IV) injection of free DOX alone, or 4 h after PLMD NP IV injection. A re-challenge study was performed by re-inoculating EMT6 cells in cured mice 120 days after the initial treatments. to validate the presence of adaptive anti-tumor immunity in the animals treated with PLMD+DOX combination, splenocyte transfer study was carried out. Ex-vivo imaging techniques were used to measure the number of infiltrated CD8+ T cells into the tumor and polarization of the M2 to M1 macrophages. Results: The PLMD+DOX combination treatment resulted in the median survival time by ~ 5.6-fold as compared to DOX alone, and led to 60% cure rate (9 out of 15 mice). This combination therapy also generated anti-tumor immunity against tumor re-inoculation in 88% of surviving mice and provided anti-tumor immunity in naive mice that received splenocyte transfer with 57% of mice without tumor growth after inoculation. Ex-vivo studies resulted a 400% increase in the number of intratumoral CD8 + T cells, 5 days after PLMD+DOX treatment, while DOX alone treatment showed only 150% increase when compared to saline treatment. Moreover, M1 macrophages-related CD86 positive cells were found up-regulated by 51% while a significant loss of M2 marker CD163 (-47%) was observed in PLMD+DOX-treated tumors compared to DOX alone-treated ones. Conclusions: The present work demonstrated that clinically suitable PLMD NPs can effectively downregulate TME-associated immunosuppression factors and boost DOX efficacy. The PLMD NPs and DOX combination therapy presents a multimodal and a translational treatment approach to enhancing DOX-induced ICD and boosting anti-tumor immunity. Citation Format: Mohammad Ali Amini, Azhar Abbasi, Ping Cai, Hoyin Lip, Jason Li, Claudia Gordijo, Li Zhang, Michael Rauth, Xiao Yu Wu. Boosting doxorubicin-induced immunogenic cell death by using tumor microenvironment modulating nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5218.

Abstract 745: Hybrid bioactive nanoparticles for modulating prostate tumor microenvironment and enhancing radiation therapy

Introduction Tumor hypoxia is a poor prognostic factor in a number of malignancies such as prostate cancer (PCa). Clinically relevant hypoxic levels are detected in 30-90% PCa with oxygen concentrations below that required for half-maximal radiosensitivity, thus making radiotherapy (RT) ineffective alone [10]. Recently our lab has created pharmaceutically acceptable bioreactive hybrid manganese dioxide nanoparticles (MDNPs) and demonstrated their ability to modulate tumor microenvironment (TME) by reacting with H 2 O 2 and protons and producing O 2 in hypoxic tumors, as well as to enhance radiation response of tumor. Methods Lipid encapsulated and polymer stabilizedMDNPs (LMD NPs) were prepared by dispersing MnO 2 precursor particles in melted lipid and polymer and then characterized for the particle size and zeta potential. In-vitro oxygen generation of LMD NPs was examined by measuring oxygen saturation level (sO 2 ) in the blood using photoacoustic (PA) imaging method with addition of LMD NPs and H 2 O 2 . Biodistribution of LMD NPs in PCa tumor-bearing mice was evaluated by using a Xenogen IVIS Spectrum Imaging System following IV injection of ICG labeled NPs through the tail vein. Mice were monitored non-invasively for up to 24 hours. To investigate the effect of combination of MDNPs and radiation therapy on tumor growth delay and survival, PCa tumor-bearing mice were treated intravenously with LMD NPs with saline as a control. Four hour post injection, 10 Gy radiation dose was given at the site of the tumor. Mice were monitored every 2 days by measuring tumor size using a caliper. Mice were sacrificed when the tumor size reached 500 mm 3 . Results PA imaging demonstrated a controlled and prolonged generation of O 2 in the blood with maximum oxygen saturation (90-95%) being reached within 60 min after incubation with LMD NPs and H 2 O 2 . The biodistribution and ex-vivo images of the resected organs showed that LMD NPs accumulated in the prostate tumor sites within 1 h post i.v. injection and remained in the tumor for at least 24 h. The ex vivo optical data of excised tissue showed a significant uptake of LMD nanoparticles by prostate tumor 24 hpost particle administration. The combination of LMD NPs treatment with single dose 10 Gy RT inhibited tumor growth by 24% at 5 days post treatment, whereas the tumor size increased about 47% in mice treated with RT alone. LMD NPs plus RT also improved survival rate of the cancerous mice for up to 53 days, which was about 3.3-fold enhancement in the mean survival rate compared to saline plus RT treatment (30 days). Conclusion The new bioreactive MnO 2 NPs exhibited desirable oxygen- generating profile and high tumor accumulation and retention after systemic administration. This work has demonstrated, in a preclinical prostate tumor model, that the combination of LMD NPs with radiation therapy is a promising treatment approach for solid tumor.. Citation Format: Mohammad Ali Amini, Azhar Z. Abbasi, Claudia R. Gordijo1, Ping Cai, Andrew M. Rauth, Robert G. Bristow, Xiao Yu Wu. Hybrid bioactive nanoparticles for modulating prostate tumor microenvironment and enhancing radiation therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 745.

A glucose-responsive insulin delivery micro device embedded with nanohydrogel particles as “smart valves”

This paper presents a glucose-responsive micro device capable of modulating drug diffusion rates according to changes in environmental glucose levels. New glucose-responsive composite membranes with enhanced mechanical strength were developed, in which chemically immobilized glucose oxidase and pH-responsive hydrogel nanoparticles were embedded within PDMS micro grids, functioning as intelligent ‘nano-valves’ in response to surrounding glucose concentration variations. Membrane responsive release profiles were quantified by testing model drug diffusion (bovine insulin), with an increase in release rates in response to increasing glucose levels. The composite membranes were integrated with PDMS drug reservoirs to form proof-of-concept devices. Release profiles of the glucose-responsive micro devices were also measured, demonstrating a marked increase in release rates of insulin when glucose levels in the surrounding media increased from 100 to 300 mg/dL.

Gadolinium-hydrogel-lipid hybrid nanoparticles provide 'off-on-off' MRI signals for non-invasive thermometry

Novel metal-chelating temperature-responsive ultrafine hydrogel nanoparticles were synthesized and loaded into a solid lipid nanoparticle carrier with various melting points. The temperature sensitive contrast agent was engineered with two consecutive ‘off-on’ and ‘on-off’ thermal transitions, which delimit a window within which thermotherapy can be applied. An ‘off-on’ step transition in contrast enhancement was detected at the melting point of the lipid matrix and an ‘on-off’ step transition is present at the lower critical solution temperature (LCST) of the copolymer hydrogel. A single composite gadolinium-hydrogel-lipid hybrid nanoparticle (Gd-HLN), as formulated here, can thus serve to indicate the onset of non-ablative thermotherapy (42-43 o C) and to delineate the transition from non-ablative to ablative temperatures (>55 o C). Since both the melting point of the lipid matrix and the LCST of the hydrogel are tunable in terms of temperature of phase transition, the intelligent multiparticle relaxation-enhancing system can be engineered to designate the temperature window specific to the goal of the individual thermotherapy.