Nanoparticles provide a unique opportunity to explore the benefits of selective distribution and release of cancer therapeutics at sites of disease through varying particle sizes and compositions that exploit the enhanced permeability of tumor-associated blood vessels. Though delivery of larger as opposed to smaller and/or actively transported molecules to the brain is prima facie a challenging endeavor, we wondered whether nanoparticles could improve the therapeutic index of existing drugs for use in treating brain tumors via these vascular effects. We therefore selected a family of nanoparticles composed of cabazitaxel-carboxymethyl cellulose amphiphilic polymers to investigate the potential for delivering a brain-penetrant taxane to intracranial brain tumors in mice. Among a small set of nanoparticle formulations, we found evidence for nanoparticle accumulation in the brain, and one such formulation demonstrated activity in an orthotopic model of glioma, suggesting that such nanoparticles could be useful for the treatment of glioma and brain metastases of other tumor types.

We report the use of flash nanoprecipitation (FNP) as an efficient and scalable means of producing Cellax nanoparticles. Cellax polymeric conjugates consisting of carboxymethyl cellulose functionalized with PEG and hydrophobic anticancer drugs, such as cabazitaxel (coined Cellax-CBZ), have been shown to have high potency against several oncology targets, including prostate cancer. FNP, a robust method used to create nanoparticles through rapid mixing, has been used to encapsulate several hydrophobic drugs with block copolymer stabilizers, but has never been used to form nanoparticles from random copolymers, such as Cellax-CBZ. To assess the potential of using FNP to produce Cellax nanoparticles, parameters such as concentration, mixing rate, solvent ratios, and subsequent dilution were tested with a target nanoparticle size range of 60 nm. Under optimized solvent conditions, particles were formed that underwent a subsequent rearrangement to form nanoparticles of 60 nm diameter, independent of Cellax-CBZ polymer concentration. This intraparticle relaxation, without interparticle association, points to a delicate balance of hydrophobic/hydrophilic domains on the polymer backbone. These particles were stable over time, and the random amphiphilicity did not lead to interparticle attractions, which would compromise the stability and corresponding narrow size distribution required for parenteral injection. The amphiphilic nature of these conjugates allows them to be processed into nanoparticles for sustained drug release and improved tumor selectivity. Preferred candidates were evaluated for plasma stability and cytotoxicity against the PC3 prostate cancer cell line in vitro. These parameters are important when assessing nanoparticle safety and for estimating potential efficacy, respectively. The optimal formulations showed plasma stability profiles consistent with long circulating nanoparticles, and cytotoxicity comparable to that of free CBZ. This study demonstrates that FNP is a promising technology for development of Cellax nanoparticles.

Effective treatment of metastatic castration resistant prostate cancer (mCRPC) remains an unmet challenge. Cabazitaxel (CBZ) is approved for mCRPC after docetaxel (DTX) failure, but the improvement in survival is only moderate (∼2 months) and patients suffer from significant side effects. Here, we report the development of a polymer based delivery system for CBZ to improve its safety and efficacy against DTX-resistant mCRPC. CBZ was conjugated to a carboxymethylcellulose-based polymer (Cellax-CBZ), which self-assembled into ∼100 nm particles in saline and exhibited sustained drug release in serum at 10%/day. Cellax-CBZ delivered 157-fold higher CBZ to PC3-RES prostate tumor in mice and could be safely administered at a 25-fold higher dose compared to free CBZ, resulting in superior tumor inhibition in multiple mice models of DTX-resistant CRPC. In a metastatic bone model of CRPC, Cellax-CBZ significantly improves overall survival with a 70% long-term survival rate to day 120, while mice treated with free CBZ had a median survival of 40 days. Cellax-CBZ induced mild and reversible neutropenia in mice but no other tissue damage. Cellax-CBZ showed significant potential for improving therapy of mCRPC over clinically approved CBZ.

Abstract Purpose: This study aimed to: (1) evaluate the antitumor efficacy of a controlled release cabazitaxel cellulose nanoparticle (CBZed-Nano) in an orthotopic bone model of docetaxel-resistant LNCaP C4-2B prostate cancer, and (2) confirm the efficacy of dose preparation by scalable instrumentation (NanoAssemblr), in a subcutaneous PC3 prostate xenograft model. Methods: mPEG-OH and cabazitaxel (CBZ) were coupled to a carboxymethylcellulose acetate polymer to yield CBZed-Nano, according to established coupling and purification methods (1). Particles were prepared by nanoprecipitation of polymer into saline by either a manual vortex technique (1) or a NanoAssemblr™ (NA) microfluidic-based instrument. Particles were purified by dialysis and sterile filtration (1). Particle size was measured by a DLS Malvern Zetasizer, morphology was screened by TEM, and residual substances were measured by LC. Male NOD-SCID mice were inoculated via intrafemoral injection with docetaxel-resistant LNCaP C42B prostate cancer. One day after inoculation, mice were treated with vortex prep CBZed-Nano (55 mg/kg) or CBZ (2 mg/kg) on a q1,5,9d schedule. Survival curves were generated (n = 10 mice per group). To directly compare preparation technique, nude mice bearing s.c. PC3 prostate cancer xenografts were treated at Bolder BioPath (Boulder, US) with CBZed-Nano (NA and vortex prep, 85 to 142 mg/kg) and CBZ (15-25 mg/kg) on a q1,5,9d schedule. Results: The CBZed-Nano polymer conjugate contained 36 wt% CBZ and 11 wt% PEG, with residual free CBZ < 0.01 wt% of CBZ content. Particles prepared by vortex and NA were 94 and 70 nm respectively (by DLS). By TEM analysis, NA particles exhibit a more uniform spherical morphology. Mice bearing docetaxel resistant LNCaP C42B tumors exhibited a partial response to CBZ treatment, with a median survival of 61 days, compared to 34 days for vehicle controls. In contrast, mice treated with CBZed-Nano exhibited a significantly improved response, with 70% durable cure. Efficacy was observed regardless of CBZed-Nano preparation technique. Mice treated with CBZ exhibited a 38-63% durable cure in the 60, 75, and 100% MTD groups. In comparison, mice treated with CBZed-Nano exhibited a 92-100% durable cure, confirming both the superior activity of CBZed-Nano relative to free CBZ, and confirming antitumor activity of the scalable NanoAssemblr production. Conclusion: The CBZed-Nano preparation technique is scalable, with manual and NanoAssemblr™ produced dose being equally efficacious in a PC3 prostate cancer model. Significant efficacy (70% durable cure) in a docetaxel-resistant prostate cancer of the bone model was also observed. The data suggests that CBZed-Nano is more tolerable than CBZ, and that it may overcome taxane-resistant solid tumor indications. (1) Bioconjugate Chem 22 2011, 2474-2486 (2) Biomaterials 33 2012, 3931-3941 Citation Format: Joseph Bteich, Bryan Hoang, Elijus Undzys, Mohammed Mohammed, Ai Lin Su, Kevin Ou, David Emerson, Kenneth Sokoll, Shyh-Dar Li, Mohit Trikha, Henry Lowman, Mark J. Ernsting. A production-scalable cabazitaxel cellulose nanoparticle exhibits significant efficacy in a bone model of docetaxel-resistant prostate cancer. [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 1342.

Pancreatic ductal adenocarcinomas are characterized by the desmoplastic reaction, a dense fibrous stroma that has been shown to be supportive of tumor cell growth, invasion, and metastasis, and has been associated with resistance to chemotherapy and reduced patient survival. Here, we investigated targeted depletion of stroma for pancreatic cancer therapy via taxane nanoparticles. Cellax-DTX polymer is a conjugate of docetaxel (DTX), polyethylene glycol (PEG), and acetylated carboxymethylcellulose, a construct which condenses into well-defined 120nm particles in an aqueous solution, and is suitable for intravenous injection. We examined Cellax-DTX treatment effects in highly stromal primary patient-derived pancreatic cancer xenografts and in a metastatic PAN02 mouse model of pancreatic cancer, focusing on specific cellular interactions in the stroma, pancreatic tumor growth and metastasis. Greater than 90% of Cellax-DTX particles accumulate in smooth muscle actin (SMA) positive cancer-associated fibroblasts which results in long-term depletion of this stromal cell population, an effect not observed with Nab-paclitaxel (Nab-PTX). The reduction in stromal density leads to a >10-fold increase in tumor perfusion, reduced tumor weight and a reduction in metastasis. Consentingly, Cellax-DTX treatment increased survival when compared to treatment with gemcitabine or Nab-PTX in a metastatic PAN02 mouse model. Cellax-DTX nanoparticles interact with the tumor-associated stroma, selectively interacting with and depleting SMA positive cells and macrophage, effects of which are associated with significant changes in tumor progression and metastasis.

Cellax, a polymer-docetaxel (DTX) conjugate that self-assembled into 120 nm particles, displayed significant enhancements in safety and efficacy over native DTX across a number of primary and metastatic tumor models. Despite these exciting preclinical data, the underlying mechanism of delivery of Cellax remains elusive. Herein, we demonstrated that serum albumin efficiently adsorbed onto the Cellax particles with a 4-fold increased avidity compared to native DTX, and the uptake of Cellax by cells was primarily driven by an albumin and SPARC (secreted protein acidic and rich in cysteine, an albumin binder) dependent internalization mechanism. In the SPARC-positive cells, a >2-fold increase in cellular internalization of Cellax was observed in the presence of albumin. In the SPARC-negative cells, no difference in Cellax internalization was observed in the presence or absence of albumin. Evaluation of the internalization mechanism using endocytotic inhibitors revealed that Cellax was internalized predominantly via a clathrin-mediated endocytotic mechanism. Upon internalization, it was demonstrated that Cellax was entrapped within the endo-lysosomal and autophagosomal compartments. Analysis of the tumor SPARC level with tumor growth inhibition of Cellax in a panel of tumor models revealed a positive and linear correlation (R(2) > 0.9). Thus, this albumin and SPARC-dependent pathway for Cellax delivery to tumors was confirmed both in vitro and in vivo.

Podophyllotoxin (PPT) exhibited significant activity against P-glycoprotein mediated multidrug resistant (MDR) tumor cell lines; however, due to its poor solubility and high toxicity, PPT cannot be dosed systemically, preventing its clinical use for MDR cancer. We developed a nanoparticle dosage form of PPT by covalently conjugating PPT and polyethylene glycol (PEG) with acetylated carboxymethyl cellulose (CMC-Ac) using one-pot esterification chemistry. The polymer conjugates self-assembled into nanoparticles (NPs) of variable sizes (20-120 nm) depending on the PPT-to-PEG molar ratio (2-20). The conjugate with a low PPT/PEG molar ratio of 2 yielded NPs with a mean diameter of 20 nm and released PPT at similar to 5%/day in serum, while conjugates with increased PPT/PEG ratios (5 and 20) produced bigger particles (30 nm and 120 nm respectively) that displayed slower drug release (similar to 2.5%/day and similar to 1%/day respectively). The 20 nm particles exhibited 2- to 5-fold enhanced cell killing potency and 5- to 20-fold increased tumor delivery compared to the larger NPs. The biodistribution of the 20 nm PPT-NPs was highly selective to the tumor with 8-fold higher accumulation than all other examined tissues, while the larger PPT-NPs (30 and 120 nm) exhibited increased liver uptake. Within the tumor, >90% of the 20 nm PPT-NPs penetrated to the hypovascular core, while the larger particles were largely restricted in the hypervascular periphery. The 20 nm PPT-NPs displayed significantly improved efficacy against MDR tumors in mice compared to the larger PPT-NPs, native PPT and the standard taxane chemotherapies, with minimal toxicity. (C) 2015 Elsevier Ltd. All rights reserved.

Docetaxel (DTX) remains the only effective drug for prolonging survival and improving quality of life of metastatic castration resistant prostate cancer (mCRPC) patients. Despite some clinical successes with DTX-based therapies, advent of cumulative toxicity and development of drug resistance limit its long-term clinical application. The integration of nanotechnology for drug delivery can be exploited to overcome the major intrinsic limitations of DTX therapy for mCRPC. We evaluated whether reformulation of DTX by facile conjugation to carboxymethylcellulose nanoparticles (Cellax) can improve the efficacy and safety of the drug in s.c. and bone metastatic models of CRPC. A single dose of the nanoparticles completely regressed s.c. PC3 tumor xenografts in mice. In addition, Cellax elicited fewer side effects compared to native DTX. Importantly, Cellax did not increase the expression of drug resistance molecules in androgen-independent PC3 prostate cancer cells in comparison with DTX. Lastly, in a bone metastatic model of CRPC, Cellax treatment afforded a 2- to 3-fold improvement in survival and enhancements in quality-of-life of the animals over DTX and saline controls. These results demonstrate the potential of Cellax in improving the treatment of mCRPC.

Abstract Tumor stroma consists of genetically normal cells that express abnormal phenotype in the tumor microenviroment, and are drivers of tumor cell proliferation and migration processes. Stromal cells can represent a significant fraction of tumor mass, and represent a barrier to drug delivery, given that stromal fibroblasts exert contractile forces which reduce vascular perfusion and increase interstitial fluid pressure. In the field, the stroma is widely recognized as a target for therapy, both to normalize the reactive microenvironment, and to reduce drug delivery barriers, but with limited clinical success. We have developed a nanoparticle delivery system for docetaxel (Cellax), a conjugate of carboxymethylcellulose and PEG, which condenses into well defined 120 nm particles suitable for intravenous administration. In addition to enhanced pharmacokinetics, 5-10X improved tumor uptake, and enhanced anti-tumor efficacy compared to approved taxanes such as native DTX (Taxotere) and nab-PTX (an albumin-paclitaxel nano-formulation, Abraxane), we have observed an anti-stromal effect in breast and pancreatic models. In a 4T1 orthotopic breast cancer model in mice, 85% of Cellax particles were taken up by cancer-associated fibroblasts (CAF), a major component of stroma. Further, in 4T1 and MDA-MB-231 breast models, Cellax treatments reduced CAF by 82 and 70% respectively, while native DTX and Nab-paclitaxel exerted no anti-stromal activity. Concomitant effects were ∼70-fold increased perfusion, ∼3-fold decreased interstitial fluid pressure (IFP), and a 7-24 fold reduction in lung metastases. In a primary human pancreatic xenograft model (OCIP19), we measured a 50% reduction in αSMA+ cells (activated pancreatic stellate cells) and macrophage within 1 day of Cellax treatment, while nab-PTX only decreased the macrophage population 3-7 days post therapy. In addition, vascular perfusion in the Cellax treated pancreatic tumor was increased 12X relative to nab-PTX and control treatment groups, with significantly reduced metastases. Preliminary mechanistic studies indicate that albumin absorbs to Cellax particles, promoting interaction with SPARC-expressing stromal cells, leading to stroma-specific activity. Citation Format: Mark Ernsting, Mami Murakami, Elijus Undzys, Shyh-Dar Li. Stromal depletion by a docetaxel nanoparticle for enhanced therapy of breast and pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4486. doi:10.1158/1538-7445.AM2014-4486

Taxanes are one of the most potent and broadest spectrum chemotherapeutics used clinically, but also induce significant side effects. Different strategies have been developed to produce a safer taxane formulation. Development of polysaccharide drug conjugates has increased in the recent years because of the demonstrated biocompatibility, biodegradability, safety, and low cost of the biopolymers. This review focuses on polysaccharide-taxane conjugates and provides an overview on various conjugation strategies and their effect on the efficacy. Detailed analyses on the designing factors of an effective polysaccharide-drug conjugate are provided with a discussion on the future direction of this field. (C) 2014 Wiley Periodicals, Inc.

UNLABELLED Taxanes are one of the most potent and broadest spectrum chemotherapeutics used clinically, but also induce significant side effects. Different strategies have been developed to produce a safer taxane formulation. Development of polysaccharide drug conjugates has increased in the recent years because of the demonstrated biocompatibility, biodegradability, safety, and low cost of the biopolymers. This review focuses on polysaccharide-taxane conjugates and provides an overview on various conjugation strategies and their effect on the efficacy. Detailed analyses on the designing factors of an effective polysaccharide-drug conjugate are provided with a discussion on the future direction of this field. For further resources related to this article, please visit the WIREs website. CONFLICT OF INTEREST The authors have declared no conflicts of interest for this article.

Taxanes are a class of anticancer agents with a broad spectrum and have been widely used to treat a variety of cancer. However, its long-term use has been hampered by accumulating toxicity and development of drug resistance. The most extensively reported mechanism of resistance is the overexpression of P-glycoprotein (Pgp). We have developed a PEGylated carboxymethylcellulose conjugate of docetaxel (Cellax), which condenses into ∼120 nm nanoparticles. Here we demonstrated that Cellax therapy did not upregulate Pgp expression in MDA-MB-231 and EMT-6 breast tumor cells, whereas a significant increase in Pgp expression was measured with native docetaxel (DTX) treatment. Treatment with DTX led to 4-7-fold higher Pgp mRNA expression and 2-fold higher Pgp protein expression compared with Cellax treatment in the in vitro and in vivo system, respectively. Cellax also exhibited significantly increased efficacy compared with that of DTX in a taxane-resistant breast tumor model. Against the highly Pgp expressing EMT6/AR1 cells, Cellax exhibited a 6.5 times lower IC50 compared with that of native DTX, and in the in vivo model, Cellax exhibited 90% tumor growth inhibition, while native DTX had no significant antitumor activity.

Nanoparticle drug delivery to the tumor is impacted by multiple factors: nanoparticles must evade clearance by renal filtration and the reticuloendothelial system, extravasate through the enlarged endothelial gaps in tumors, penetrate through dense stroma in the tumor microenvironment to reach the tumor cells, remain in the tumor tissue for a prolonged period of time, and finally release the active agent to induce pharmacological effect. The physicochemical properties of nanoparticles such as size, shape, surface charge, surface chemistry (PEGylation, ligand conjugation) and composition affect the pharmacokinetics, biodistribution, intratumoral penetration and tumor bioavailability. On the other hand, tumor biology (blood flow, perfusion, permeability, interstitial fluid pressure and stroma content) and patient characteristics (age, gender, tumor type, tumor location, body composition and prior treatments) also have impact on drug delivery by nanoparticles. It is now believed that both nanoparticles and the tumor microenvironment have to be optimized or adjusted for optimal delivery. This review provides a comprehensive summary of how these nanoparticle and biological factors impact nanoparticle delivery to tumors, with discussion on how the tumor microenvironment can be adjusted and how patients can be stratified by imaging methods to receive the maximal benefit of nanomedicine. Perspectives and future directions are also provided.

The majority of ultrafast temperature sensitive liposome (uTSL) formulations reported in the literature deliver the highly membrane permeable drug, doxorubicin (DOX). Here we report on the study of the uTSL formulation, HaT (Heat activated cytoToxic, composed of the phospholipid DPPC and the surfactant Brij78) loaded with the water-soluble, but poorly membrane permeable anticancer drugs, gemcitabine (GEM) and oxaliplatin (OXA). The HaT formulation displayed ultrafast release of these drugs in response to temperature, whereas attempts with LTSL (Lyso-lipid Temperature Sensitive Liposome, composed of DPPC, MSPC, and DSPE-PEG) were unsuccessful. HaT-GEM and HaT-OXA both released >80% of the encapsulated drug within 2 min at 40-42 °C, with <5% drug leakage at 37 °C after 30 min in serum. The pharmacokinetic profile of both drugs was improved by formulating with HaT relative to the free drug, with clearance reduced by 50-fold for GEM and 3-fold for OXA. HaT-GEM and HaT-OXA both displayed improved drug uptake in the heated tumor relative to the unheated tumor (by 9-fold and 3-fold, respectively). In particular, HaT-GEM showed 25-fold improved delivery to the heated tumor relative to free GEM and significantly enhanced antitumor efficacy with complete tumor regression after a single dose of HaT-GEM. These data suggest that uTSL technology can also be used to deliver nonmembrane permeable drugs via an intravascular ultrafast release mechanism to great effect.

Chapter 11 Theranostic Nanoparticles for Cancer Imaging and Therapy Mami Murakami, Mami MurakamiSearch for more papers by this authorMark J. Ernsting, Mark J. ErnstingSearch for more papers by this authorShyh-Dar Li, Shyh-Dar LiSearch for more papers by this author Mami Murakami, Mami MurakamiSearch for more papers by this authorMark J. Ernsting, Mark J. ErnstingSearch for more papers by this authorShyh-Dar Li, Shyh-Dar LiSearch for more papers by this author Book Editor(s):Ashutosh Tiwari, Ashutosh TiwariSearch for more papers by this authorAtul Tiwari, Atul TiwariSearch for more papers by this author First published: 04 February 2013 https://doi.org/10.1002/9781118644591.ch11Citations: 3 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary This chapter contains sections titled: Introduction Multifunctional Nanoparticles for Noninvasive Monitoring of Biodistribution Multifunctional Nanoparticles for Monitoring Drug Release Theranostics to Image Therapeutic Response Conclusion and Future Directions Citing Literature Nanomaterials in Drug Delivery, Imaging, and Tissue Engineering RelatedInformation

Docetaxel-conjugate nanoparticles, known as Cellax, were synthesized by covalently conjugating docetaxel and polyethylene glycol to acetylated carboxymethylcellulose via ester linkages, yielding a polymeric conjugate that self-assembled into 120 nm particles suitable for intravenous administration. In 4T1 and MDA-MB-231 orthotopic breast tumor models, Cellax therapy reduced α-smooth muscle actin (α-SMA) content by 82% and 70%, respectively, whereas native docetaxel and nab-paclitaxel (albumin-paclitaxel nanoparticle, Abraxane) exerted no significant antistromal activity. In Cellax-treated mice, tumor perfusion was increased by approximately 70-fold (FITC-lectin binding), tumor vascular permeability was enhanced by more than 30% (dynamic contrast-enhanced magnetic resonance imaging), tumor matrix was decreased by 2.5-fold (immunohistochemistry), and tumor interstitial fluid pressure was suppressed by approximately 3-fold after Cellax therapy compared with the control, native docetaxel, and nab-paclitaxel groups. The antistromal effect of Cellax treatment corresponded to a significantly enhanced antimetastatic effect: lung nodules were reduced by 7- to 24-fold by Cellax treatment, whereas native docetaxel and nab-paclitaxel treatments were ineffective. Studies of the 4T1 tumor showed that more than 85% of the Cellax nanoparticles were delivered to the α-SMA+ stroma. Significant tumor stromal depletion occurred within 16 hours (∼50% depletion) postinjection, and the α-SMA+ stroma population was almost undetectable (∼3%) by 1 week. The 4T1 tumor epithelial cell population was not significantly reduced in the week after Cellax injection. These data suggest that Cellax targets tumor stroma and performs more efficaciously than docetaxel and nab-paclitaxel.

SUMMARY Cellax is a polymer conjugate of docetaxel (DTX) which self-assembles into well-defined 120 nm nanoparticles in saline. This delivery system extends blood circulation, increases tumor uptake, and interacts preferentially with stromal cells. In a 4T1 orthotopic breast cancer model in mice, 85% of Cellax particles were taken up by cancer-associated fibroblasts (CAF), a major component of stroma. Further, Cellax treatments reduced CAF by 82%, while native DTX and Nab-paclitaxel exerted no anti-stromal activity. Concomitant effects were ~70fold increased perfusion, ~3-fold decreased interstitial fluid pressure (IFP), and a 7-fold reduction in lung metastases. Taken together, the data suggests that stromal depletion enhances control of tumor growth and metastases, and that stromal control can be an important component of therapy. INTRODUCTION Nanoparticle delivery of drugs can enhance blood circulation and tumor uptake, and reduce toxicity by limiting non-selective uptake. Increasingly, investigators are addressing particle fate in the tumor: drug must be released or interact with the target cells in order to have an effect. We designed a drug delivery system around a carboxymethylcellulose scaffold, in which a high hydrophobic DTX content (37 wt%) and a PEG component (5 wt%) drive self-assembly into a defined nanoparticle (120 nm), that extends blood circulation, enhances tumor uptake, and is readily internalized by cells (1-4). The Cellax therapeutic demonstrates significant enhancements to efficacy and safety, relative to approved taxane therapies (14). Here we report on the unique therapeutic impact of Cellax in the tumor microenvironment compared to native DTX and Nab-paclitaxel (Abraxane). EXPERIMENTAL METHODS Cellax polymer was synthesized as previously reported (1): carboxymethylcellulose was converted to a free acid and acetylated, followed by EDC coupling of DTX and PEG, to yield a polymer with 37.1 +/1.5 wt% DTX and 4.7 +/0.8 wt% PEG (Fig 1). Cellax particles were prepared by precipitation of acetonitrile solutions of polymer into 0.9% saline, followed by dialysis, concentration, and sterile filtration. Dose was determined by UV measurement. Figure 1: Depiction of the Cellax polymer Animal studies: The experimental protocols in this study were approved by the Animal Care Committee of the University Health Network (UHN, Toronto, ON, Canada). DTX was formulated in a Tween80/ethanol/saline (20:13:67) solution. Nabpaclitaxel (Abraxane) was sourced from the UHN pharmacy. Orthotopic 4T1 models: 4T1 cells (1x10) were inoculated to the mammary fat pad of female Balb/c mice (n=10 per group). Intratumoral distribution study: when 4T1 tumors reached 4-5 mm in diameter, mice were treated with Cellax (170 mg DTX/kg) and were sacrificed 6, 16, 24, 72 and 168 h after treatment. Fixed tumor sections were stained for α-SMA+ CAF and with H&E, and images were analyzed by Definiens Tissue Studio software for total tumor area, non-viable component, and stromal coverage. In a related study, mice were treated with Cellax containing fluorescent DiI, were sacrificed 24h later, and the fixed tumors were stained for αSMA+ CAF. Tumor sections were imaged and colocalization of CAF with Cellax-DiI was calculated in Definiens software. Efficacy model: when tumors reached 5-7 mm, mice were treated with the MTD of Cellax (170 mg DTX/kg), native DTX (40 mg DTX/kg), Nab-paclitaxel (50 mg PTX/kg) or saline via tail vein injection. Six days post therapy, mice were injected with FITC-lectin, tumor interstitial fluid pressure (IFP) was measured, and the primary tumors were then resected and fixed for histology. Tumor sections were stained for CD31 (blood vessels) and for α-SMA (CAF). On day 13, mice were treated with a second round of therapy, and on day 20 the entire cohort was sacrificed, and lung tissues were harvested for histology (H&E staining, quantification of tumor burden with

SUMMARY Thermosensitive liposomes (TSLs) provide a triggered delivery system for the localized delivery of chemotherapeutic drugs to tumors. This technology is showing potential in late stage clinical trials for recurrent breast cancer and hepatocellular carcinoma (ThermoDox, Celsion Inc.), and if successful this could lead to an increased demand for these types of treatments in the clinic. TSLs have generally been applied to membrane permeable drugs, such as doxorubicin (DOX), and it seems this concept has not yet been studied extensively for formulations of other less membrane permeable drugs. HaT is a novel TSL formulation developed by us, and here we describe our findings using it for the delivery of gemcitabine (GEM) and oxaliplatin (OXA). Pharmacokinetic (PK), biodistribution and efficacy data has been studied and the key findings are discussed. INTRODUCTION The delivery of a drug to its target with minimal systemic toxicity is a major goal of drug delivery research. Through the use of TSLs in combination with advanced image-guided heating methods, the potential of specific and localized delivery is becoming more of a reality. TSLs are designed to release their payload at mildhyperthermic temperatures (39-43°C). For optimal performance this heating is applied to just the tumor target, leaving the rest of the vasculature at normal physiological temperature. In this way, the TSL remains stable at 37°C as it passes through the systemic circulation and the drug is protected by the liposomal membrane allowing increased drug to flow through the bloodstream by minimizing clearance and non-specific uptake. When the TSL reaches the heated tumor (3943°C), it releases its contents rapidly, causing a high local concentration of drug within the vasculature of the tumor. At these high concentrations the drug penetrates into the tumor and can act via understood mechanisms to bring about a therapeutic response. This concept of TSL mediated drug delivery has been studied previously and is now being investigated in clinical trials for a number of different cancer indications. The trailblazing formulation of this type is LTSL (Lysolipid Temperature Sensitive Liposome, DPPC/ MSPC/DSPE-PEG2000, 86/10/4 mol%) also known as ThermoDox, which will reach the end of a phase III trial for hepatocellular carcinoma later this year (www.clinicaltrials.gov, ID: NCT00617981; other clinical trials for this formulation are also ongoing). To date, LTSL and other therapeutic TSL formulations have mainly been studied for the delivery of DOX, a highly membrane permeable cytotoxic. However, DOX is a known cardio toxin, and lifetime doses of DOX are therefore limited. For this reason we are interested in developing TSLs suitable for the delivery of other drugs which will not compound the problem of potential DOX cardiotoxicity and will also be suitable for DOX-resistant indications. In particular, we have chosen GEM and OXA as drugs to investigate for delivery via a TSL mediated strategy. Rather than use LTSL, we have developed a simpler 2 component TSL called HaT (Heat activated cytoToxic, DPPC/Brij78, 96:4 mol%). Originally this formulation was developed to deliver DOX, which showed faster drug release at mild-hyperthermic temperatures and improved drug stability at 37-38°C compared to LTSL. These improvements led to further enhanced tumoral drug uptake and efficacy in vivo relative to LTSL. HaT has been used here to deliver GEM or OXA via a burst release action in heated tumors. EXPERIMENTAL METHODS LTSL was formulated with 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC), 1-stearoyl-2-hydroxy-snglycero-3-phosphatidylcholine (MSPC) and 1,2distearoyl-sn-glycero-3-phosphatiylethanol-amine-N[methoxy (polyethyleneglycol)-2000] (DSPE-PEG2000) in a 86/10/4 molar ratio. HaT was formulated with DPPC and Brij78 (polyethoxyethylene stearyl ether) in a 96/4 molar ratio. All liposomes were prepared by thin film hydration with a ~10 mg/mL solution of drug (GEM or OXA) in buffer. Each formulation was extruded to size, and the outer phase was exchanged with buffer by dialysis. The drug concentration was determined after the disruption of liposomes with Triton X-100. Drug concentrations were analyzed by HPLC-MS (GEM) and ICP-AES (OXA) methods. Encapsulation efficiency of all passively loaded drugs was ~10%. Drug release models: Liposomes (~200 μg/mL Drug) in release buffer were incubated at different temperatures (37-42°C) for given times, then immediately put on ice. Samples were diluted with buffer, filtered, and analyzed for released drug (HPLC-MS or ICP-AES). In vivo studies: female BALB/c mice (aged 5-6 weeks, 18-20 g) were purchased from The Jackson Laboratory (Bar Harbor, ME). All experimental protocols in this study were approved by the Animal Care Committee of the University Health Network (Toronto, Ontario, Canada) in accordance with the policies established in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council of Animal Care. Briefly, PAN02 or EMT-6 cells were s.c. implanted into a leg of C57BL6 or BALB/c mice respectively, and 7 days post-tumor inoculation (tumor mass was approximately 0.2-0.3 g), the mice were anesthetized, and the tumor bearing leg was immersed in a water bath maintained at 43°C. The tumor was heated for 10 min for temperature equilibration before the i.v. injection of different drug formulations, and then heating for a further hour. For the biodistribution study, blood, heart, kidney, liver, lung, spleen and tumors were harvested following the treatment. For studies with GEM the tissue was homogenized and an aliquot of the supernatant was studied by HPLC-MS. For OXA experiments, the samples were digested with concentrated acid and then studied for Pt concentration by ICP-AES. For the efficacy study, PAN02 or EMT-6 tumorbearing mice were treated as above, and tumor size and body weight were monitored until endpoint. RESULTS AND DISCUSSION HaT-GEM and LTSL-GEM formulations were prepared and showed similar physical characteristics (size ~100 nm, PDI < 0.1). We encountered problems preparing formulations of LTSL with OXA solutions using the passive hydration method. Investigations into the cause of this are ongoing, but at present we believe it may be related to the incompatibility of the LTSL lipid composition and the low NaCl concentration necessary for working with OXA (5% dextrose was the buffer used). For this reason, no comparisons between HaT-OXA and its LTSL counterpart could be made. HaT formulations of OXA were formed in the usual way. HaT-GEM released >80% GEM within 1 min at 4142°C, with <5% drug leakage at 37°C after 30 min in serum, while LTSL-GEM was stable at 37°C but exhibited slower release at 41°C (1%) and 42°C (~70%). This can be represented as 1.5 to 8-fold decreased release rate constants at 41-42°C for LTSL relative to HaT under the same conditions. Figure 1: Summary of efficacy endpoints for HaT-GEM and HaT-OXA formulations in combination with localized hyperthermia compared to free drug and control. The PK profile of GEM was improved for both liposomal formulations with ~100% injected dose (ID) left in the blood after 1 h and ~9% remaining after 4 h. This was compared to ~ 1% and 0.03% ID respectively for free GEM. HaT-GEM improved drug delivery to the heated tumor relative to LTSL-GEM by 7.5-fold, and significantly enhanced antitumor efficacy with complete inhibition of tumor growth after a single dose of HaTGEM (Figure 1a). The release rates of HaT-OXA were rapid at 4142°C, with >80% released within the first minute, but <5% release at 37°C. HaT-OXA displayed an enhanced retention of drug in the blood with ~ 15% ID after 1 h compared to ~2% for free OXA. Antitumor efficacy was considerably improved relative to free drug, with minimal tumor growth after 7 days following a single dose (Figure 1b). CONCLUSION Here we describe the successful formulation of HaTGEM and HaT-OXA TSLs. Where possible we have compared the HaT formulations with LTSL formulations of the same drug. The HaT formulations display improved PK, increased drug uptake and improved efficacy relative to the free drugs. These formulations may provide a useful treatment for situations where DOX is either ineffective or where patients have reached maximum exposure levels of DOX already. REFERENCES 1. Grull, S.; Langereis, S.; J. Control. Release 2012, 161, 317-327 2. May, J.P.; Li, S.D.; Recent Patents on Biomedical Engineering 2012, 5, 148-158 3. Needham, D.; Anyarambhatla, G.; Kong, G.; Dewhirst M.W.; Cancer Res. 2000, 60, 1197-1201 4. Landon, C.; Park, C.J.; Needham, D.; Dewhirst M.W.; The Open Nanomedicine Journal 2011, 3, 3864 5. Tagami, T.; Ernsting, M.J.; Li, S.D.; J. Control. Release 2011, 152, 303-309 6. Tagami, T. Ernsting, M.J.; Li, S.D.; J. Control. Release 2011, 154, 290-297 7. Tagami, T.; Foltz, W.D.; Ernsting, M.J.; Lee, C.M.; Tannock, I.F.; May, J.P.; Li, S.D.; Biomaterials 2011, 32, 6570-6578 8. Tagami, T.; May, J.P.; Ernsting, M.J.; Li, S.D.; J. Control. Release 2012, 161, 142-149

Here we report the development of an enhanced thermosensitive formulation composed of DPPC and Brij78, loaded with doxorubicin (DOX) using a Cu2+ gradient and post-inserted with an additional amount of Brij78. This optimal formulation (HaT-II: Hyperthermia-activated cytoToxic) displayed significantly improved stability in serum at 37 °C, and enhanced drug release rates at 41–42 °C, compared to LTSL (lyso-lipid temperature sensitive liposomes, DPPC/MSPC/DSPE-PEG2000 = 86/10/4, pH gradient drug loading). HaT-II released 100% DOX within 15–40 s at 40–42 °C, with only 5% drug leakage at 37 °C after 30 min in serum, while LTSL lost 30% of its drug content at 37 °C and exhibited ~ 2-fold decreased release rate constants at 41–42 °C under the same conditions. The pharmacokinetics of DOX was significantly improved in non-heated HaT-II treated healthy mice with 2.5-fold increased area under the curve and 2-fold prolonged circulation half life compared to LTSL. This led to 2-fold improved drug delivery to the heated tumor by HaT-II (~ 20% injected dose/g tissue), relative to LTSL and significantly enhanced antitumor efficacy with complete inhibition of tumor growth after a single dose of HaT-II. Finally, HaT-II exhibited little toxicity in mice, inducing no body weight loss and no abnormality in the blood chemistry (10 mg DOX/kg).

Cellax is a PEGylated carboxymethylcellulose conjugate of docetaxel (DTX) which condenses into a 120-nm nanoparticle, and was compared against the approved clinical taxane nanoformulation (Abraxane®) in mouse models. Cellax increased the systemic exposure of taxanes by 37× compared to Abraxane, and improved the delivery specificity: Cellax uptake was selective to the tumor, liver and spleen, with a 203× increase in tumor accumulation compared to Abraxane. The concentration of released DTX in Cellax treated tumors was well above the IC50 for at least 10 d, while paclitaxel released from Abraxane was undetectable after 24h. In s.c. PC3 (prostate) and B16F10 (melanoma) models, Cellax exhibited enhanced efficacy and was better tolerated compared to Abraxane. In an orthotopic 4T1 breast tumor model, Cellax reduced the incidence of lung metastasis to 40% with no metastasic incidence in other tissues. Mice treated with Abraxane displayed increased lung metastasic incidence (>85%) with metastases detected in the bone, liver, spleen and kidney. These results confirm that Cellax is a more effective drug delivery strategy compared to the approved taxane nanomedicine.