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Differences in Nanoparticle Uptake in Transplanted and Autochthonous Models of Pancreatic Cancer Zhimin Tao, Mandar Deepak Muzumdar, Alexandre Detappe, Xing Huang, Eric Xu, Yingjie Yu, Tarek H Mouhieddine, Haiqin Song, Tyler Jacks, and P. Peter Ghoroghchian Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b04043 • Publication Date (Web): 13 Mar 2018 Downloaded from http://pubs.acs.org on March 14, 2018
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Differences in Nanoparticle Uptake in Transplanted and Autochthonous Models of Pancreatic Cancer Zhimin Tao,1,* Mandar Deepak Muzumdar,1,2,3,4* Alexandre Detappe,1,2,3 Xing Huang,1 Eric S. Xu,1 Yingjie Yu,1 Tarek H. Mouhieddine,2,3 Haiqin Song,1 Tyler Jacks,1 and P. Peter Ghoroghchian1,2,3,#
1. Koch Institute for Integrative Cancer Research at MIT, 500 Main Street, Cambridge, MA 02139, USA 2. Dana Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA 3. Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA 4. Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA * Denotes equal author contribution
# To whom correspondence should be addressed:
P. Peter Ghoroghchian, M.D., Ph.D. Charles W. and Jennifer C. Johnson Clinical Investigator Koch Institute for Integrative Cancer Research Massachusetts Institute of Technology 77 Massachusetts Avenue, 76-261F Cambridge, MA 02139, USA (617) 252-1163
[email protected] 1 ACS Paragon Plus Environment
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ABSTRACT
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Human pancreatic ductal adenocarcinoma (PDAC) contains a distinctively dense stroma that
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limits the accessibility of anticancer drugs, contributing to its poor overall prognosis. Nanoparticles
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can enhance drug delivery and retention in pancreatic tumors and have been utilized clinically for
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their treatment. In preclinical studies, various mouse models differentially recapitulate the
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microenvironmental features of human PDAC. Here, we demonstrate that through utilization of
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different organic co-solvents and by doping of a homopolymer of poly(-caprolactone), a diblock
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copolymer composition of poly(ethylene oxide)-block-poly(-caprolactone) may be utilized to
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generate biodegradable and nanoscale micelles with different physical properties. Noninvasive
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optical imaging was employed to examine the pharmacology and biodistribution of these various
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nanoparticle formulations in both allografted and autochthonous mouse models of PDAC. In
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contrast to the results reported with transplanted tumors, spherical micelles as large as 300 nm
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in diameter were found to extravasate in the autochthonous model, reaching a distance of
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approximately 20 m from the nearest tumor cell clusters. A lipophilic platinum(IV) prodrug of
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oxaliplatin was further able to achieve a ~7-fold higher peak accumulation and a ~50-fold increase
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in its retention half-life in pancreatic tumors when delivered with 100 nm-long worm-like micelles
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as when compared to the free drug formulation of oxaliplatin. Through further engineering of
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nanoparticle properties, as well as by widespread adoption of the autochthonous tumor model for
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preclinical testing, future therapeutic formulations may further enhance the targeting and
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penetration of anticancer agents to improve survival outcomes in PDAC.
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KEYWORDS
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Drug delivery; Nanomedicine; Pancreatic ductal adenocarcinoma; Platinum(IV); Optical Imaging
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Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United
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States and a significant contributor to morbidity and mortality worldwide1, 2. In spite of advances
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in combination chemotherapy (e.g., FOLFIRINOX3 and gemcitabine/nab-paclitaxel4), which offer
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greater response rates and prolong median survival, long-term survival remains poor at 8%1, 2.
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These therapies also induce considerable adverse effects, limiting their use to patients with good
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performance status. Given that most patients are diagnosed with unresectable disease at the time
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of initial presentation2, novel approaches for the treatment of advanced pancreatic cancers are
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desperately needed.
32 33
A defining feature of PDAC is the extraordinary heterogeneity of the tumor microenvironment5-7.
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Pancreatic tumor cells are surrounded by a dense stroma that possesses diverse cellular (e.g.,
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fibroblasts, macrophages, immune cells) and acellular components (e.g., extracellular matrix
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proteins, hyaluronic acid), which together can comprise > 90% of the tumor by volume6. These
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stromal features contribute to increased interstitial pressures, impairing intratumoral blood flow
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and therapeutic delivery to PDAC cells. Efforts to modify the cellular (e.g., through hedgehog
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inhibition8) and acellular microenvironment (e.g., with the use of hyaluronidase9, 10) have improved
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vascularization and tumor responses in preclinical PDAC models; they have also shown early
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promise in clinical trials11, 12. Overcoming the stromal barrier can, thus, enhance the efficacy of
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existing cytotoxic chemotherapies directed against PDAC, potentially affording greater tumor
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responses at lower doses and with fewer systemic side effects.
44 45
Nanomedicine offers a means by which to improve anticancer drug delivery through both passive
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and active targeting mechanisms that promote nanoparticle accumulation in tumors13-16.
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Nanoparticles have also been successfully utilized to improve clinical outcomes in pancreatic
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cancers4, 17. Conjugation of cancer cell-specific targeting moieties to nanoparticles can potentially
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promote greater drug accumulation at tumor sites, minimizing side effects and maximizing 3 ACS Paragon Plus Environment
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therapeutic activity18-20. While lipid-based nanoparticles have been widely-adopted in translational
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research due to their outstanding biocompatibility, their labile nature may affect stability during
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both product storage and in vivo administration; polymeric nanoparticles can be better engineered
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for controlled biodegradability but must overcome concerns related to potential toxicity and
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immunogenicity.21-26 As an example of the successful preclinical application of the latter, platinum-
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based anticancer agents have been incorporated into polymeric micelles to prolong their blood
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circulation times and to enhance tumor accumulation in transgenic as well as in transplanted
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models of human PDAC27-30. These studies have revealed the potential for nanoparticles of
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smaller sizes to exhibit preferential accrual in pancreatic tumors28, 31. Little is known, however,
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about the effects of other nanoparticle characteristics, such as shape, on affecting drug delivery.
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Moreover, the inconsistent findings reported from previous nanoparticle studies may be
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attributable to differences in vascular density, in the sizes of endothelial gap junctions, and in the
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extent of the desmoplastic stroma in the specific transgenic or transplanted tumor model that was
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utilized; these key features play critical roles in disease pathogenesis, in limiting nanoparticle
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extravasation and parenchymal drug diffusion, and in stymying the efficacy of antitumor therapies.
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It is important to underscore that all transgenic and transplanted tumor models fail to faithfully
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recapitulate the genetic and biological characteristics of human PDAC.8
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Genetically-engineered mouse models of pancreatic cancer have been recently developed in
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which tumors arise spontaneously in the mouse’s own pancreas (i.e., autochthonous models)32-
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34.
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wise progression of human PDAC, including the development of its desmoplastic stroma32-34. We,
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therefore, utilized both subcutaneous transplant and autochthonous murine models of PDAC to
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systematically evaluate the interplay between different nanoparticle characteristics (e.g., size and
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shape) and their impact on overcoming the unique features of each tumor model (i.e., differences
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in vascular and stromal density) to augment in vivo delivery.
These models accurately mimic the genetic and molecular changes that occur during the step-
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Generation of polymeric nanoparticles of varying size and shape for in vivo delivery
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Poly(ethylene oxide)-block-poly(-caprolactone) (PEO-b-PCL) is an amphiphilic diblock
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copolymer composition comprised of two FDA-approved building blocks that impart favorable
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physicochemical and biological characteristics, including prolonged in vivo stability, enhanced
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aqueous solubility (imparted by the PEO block), full biodegradability (of the PCL block), and low
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in vitro or in vivo toxicity; moreover, while repeated administration of any PEO-coated nanoparticle
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may elicit a humoral immune response, PEO-b-PCL-based vehicles have been shown to induce
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minimal immunogenicity.35 We have previously explored the phase behavior of different PEO-b-
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PCL molecular weight compositions, identifying those that readily generate nanoscale vesicles
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and spherical micelles in aqueous suspension36, 37. We have demonstrated that PEO-b-PCL-
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based vesicles can incorporate both chemotherapeutic species36 and near-infrared (NIR)
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fluorophores38. Additionally, we have conducted in vivo optical imaging studies that have
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determined preferential accumulation of PEO-b-PCL-based spherical nanoparticles within
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peritoneal tumor implants after intraperitoneal as opposed to intravenous (IV) administration 39.
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Previous investigations have also reported that PEO-b-PCL-based worm-like micelles (as
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opposed to nanospheres) may achieve enhanced penetration through dense tumor tissues40, 41.
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Here, we sought to explore the size and shape dependence of PEO-b-PCL-based nanoparticles
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to accumulate in pancreatic tumors in hopes of identifying key physical characteristics that may
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enhance drug delivery. While other studies have explored the size dependencies of nanoparticles
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to accumulate in pancreatic tumor xenografts28, the Kras- and p53-mutant genetically-engineered
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mouse model (KPC; see Materials and Methods) more readily recapitulates the key genetic,
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histologic, and microenviromental features of PDAC33, 34, 42, 43 (Figure 1A, 1B and Supplementary
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Figure S1). Previous work revealed that transplant and autochthonous PDAC models display
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marked differences in drug delivery. Specifically, transplant tumors show greater responses to 5 ACS Paragon Plus Environment
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chemotherapy and increased perfusion compared to the autochthonous PDAC model. Further,
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both human and mouse transplanted tumors exhibit greater vascularity (i.e., greater blood vessel
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numbers and smaller vessel-to-tumor cell distances) than their naturally arising counterparts.8 As
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such, we sought to conduct a comparative study of nanoparticle delivery between the
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autochthonous KPC and traditional transplant models.
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We first focused on identifying fabrication conditions that could give rise to nanoparticles that
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differed with respect to size and shape, utilizing two PEO-b-PCL compositions (PEO5k-PCL10k and
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PEO5k-PCL16k), a homopolymer of PCL30k, and two lipophilic encapsulants that could be loaded
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within the resultant nanoparticles (Figure 1C). The near-infrared (NIR-) emissive carbocyanine
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dye DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) was selected as a
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model imaging agent and a novel lipophilic prodrug of oxaliplatin (C16-oxaliplatin(IV)-C16) was
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synthesized for nanoparticle-based encapsulation and delivery to pancreatic tumors.
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Encapsulation of lipophilic DiR in PEO-b-PCL-based nanoparticles has previously been used to
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enable their accurate in vivo imaging in subcutaneous tumor xenograft models.44, 45 We have also
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utilized DiR that was encapsulated in PEO-b-PCL-wrapped lanthanide nanoparticles to
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successfully image and compare the extent of tumor localization after either intraperitoneal or
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intravenous administration into a disseminated cell-line xenograft model of ovarian cancer,
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validating tumor cell (delineated by emission of red fluorescent protein, RFP) and nanoparticle
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biodistribution (demarcated by the non-overlapping NIR emission of DiR) in vivo as well as ex
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vivo both in whole-excised organs and in tissue sections via microscopy.39
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To generate nanoparticles of varying size and shape for comparative analyses, we dissolved the
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PEO-b-PCL copolymers with and without the PCL homopolymer and/or the lipophilic
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encapsulants (i.e., DiR and/or C16-oxaliplatin(IV)-C16) into two different organic solvents (Figure
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1D, Supplementary Scheme S1 and Materials and Methods). When dissolved in THF (and
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subsequently diluted into larger aqueous volumes), PEO-b-PCL diblock copolymers produced
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suspensions of spherical nanoparticles; polymers of higher molecular weight formed
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nanoparticles of larger size as assessed by cryogenic transmission electron microscopy (cryo-
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TEM; Figure 2A). Interestingly, by first dissolving in water-miscible DMF (followed by aqueous
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dilution), the same PEO-b-PCL compositions led to the formation of small worm-like micelles. To
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our knowledge, this is the first example by which different shaped nanoparticles are produced
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from the same diblock copolymer compositions simply by changing the miscible organic solvent
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used for aqueous emulsion. Dynamic Light Scattering (DLS) revealed that the spherical
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nanoparticles made from PEO5k-PCL10k, PEO5k-PCL16k, and PEO5k-PCL16k:PCL30k (1:1 molar
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ratio) possessed hydrodynamic diameters of ~70 nm, ~100 nm, and ~300 nm, respectively. In
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contrast, worm-like micelles made from PEO5k-PCL10k had a mean length of 100 nm. We
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confirmed the stability of each PEO-b-PCL-based formulation; sizes (Figure 2B), polydispersity
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indexes (PDIs) (Figure 2C), and zeta potential measurements (Figure 2D) from all nanoparticle
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suspensions were relatively constant over the course of one week. Their surface charges were
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mildly negative and were likely attributable to their neutrally-charged PEO shells. Through the
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selection of different initial molecular weight compositions and the simple exchange of organic
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solvents used for aqueous emulsion, we were, thus, able to reproducibly generate stable
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polymeric nanoparticles of varying size and shape for further in vitro and in vivo experimentation.
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Prior to their use for in vivo delivery of anticancer agents, we first examined the in vitro cytotoxicity
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of the various PEO-b-PCL-based nanoparticle formulations. We treated Kras-mutant B22 murine
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PDAC cells (see Materials and Methods) with different concentrations of PEO-b-PCL-based
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nanoparticles of varying size and morphology and measured in vitro cell viability at 72 h
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(Supplementary Figure S2). The IC50 values were determined to be 10, 17.8 and 177.8 mg/mL
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(corresponding to 0.5, 0.7, and 11.9 mM of polymer) for PEO5k-PCL16k (THF), PEO5k7 ACS Paragon Plus Environment
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PCL16k/PCL30k (THF), and PEO5k-PCL10k (DMF) nanoparticles, respectively. The cytotoxicity of
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each nanoparticle formulation correlated with its decreasing size and spherical shape: the toxicity
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imparted by PEO5k-PCL16k (THF; 100 nm diameter spherical micelles) > PEO5k-PCL16k/PCL30k
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(THF; 300 nm-diameter spherical micelles) > PEO5k-PCL10k (DMF; 100 nm-length worm-like
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micelles). The worm-like micelles displayed the lowest toxicity on B22 cells as compared to each
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spherical nanocarrier. While smaller particles may have been expected to introduce slightly more
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cytotoxicity (at a given polymer concentration) due to improvements in intracellular uptake, the
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dramatic differences in cytotoxicity between nanoparticles of different shape but of the same
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overall dimensions was not expected and warrants further study. Nevertheless, these data
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confirmed that the PEO-b-PCL-based nanoparticles were suitably non-toxic for in vivo use.
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To facilitate in vivo optical imaging of PEO-b-PCL-based nanoparticles, we loaded them with the
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NIR fluorophore DiR (see Materials and Methods). We observed linear concentration-dependent
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changes in the fluorescence emission (Figure 3A) and the solution absorption of DiR (Figure 3B
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and Supplementary Figure S3) when encapsulated in PEO5k-PCL16k–based nanospheres in
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water. With increasing DiR concentrations, emission and absorbance initially rose but then
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declined (Figure 3A and 3B), likely due to the self-quenching effect and solubility limit,
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respectively, of DiR confined in the polymer shell. Consistent with the lowered energy states
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imparted by the self-quenching and stacking of dye molecules, the peak emission wavelength of
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DiR displayed a bathochromic shift with increasing concentration in nanoparticle suspensions. To
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minimize self-quenching, we adopted an initial solution concentration of DiR equal to 5 µM for
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generation of all nanoparticles used for subsequent in vivo studies. To ensure that this loading
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concentration enabled linear concentration-dependent detection of particles by in vivo imaging,
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we next administered DiR-encapsulated PEO5k-PCL16k–based nanospheres at different
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concentrations to nude mice by IV tail-vein injection. We subsequently measured the fluorescence
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intensities values of the particles in blood. We observed an increasing linear correlation between 8 ACS Paragon Plus Environment
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fluorescence efficiency and the DiR concentration at the administered dose level (Figure 3C). We
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also measured the effects of DiR loading on the chemical properties of all the various nanoparticle
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formulations. While their final concentrations of encapsulated DiR varied by as much as 2-fold in
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different solutions (Table 1), the DiR-loaded nanoparticles were highly photostable in water, in
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phosphate-buffered saline (PBS), and in fetal-bovine serum (FBS) (Figure 3D).
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Delivery of PEO-b-PCL-based nanoparticles to allografted PDAC tumors in mice
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We evaluated whether the efficiency of tumor accumulation of our PEO-b-PCL-based
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nanoparticles depended on their size and/or shape, using an allografted murine model of PDAC.
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We transduced B22 Kras; p53 mutant murine PDAC cells derived from a mouse model that closely
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recapitulates the human disease46 with a retroviral construct, permitting expression of both
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luciferase
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immunofluorescence analysis of tumor sections). We transplanted these cells into the flanks of
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immunocompromised nude mice, allowing for subcutaneous tumor growth, and then administered
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various DiR-encapsulated, PEO-b-PCL-based nanoparticle formulations via IV tail-vein injection.
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The nanoparticles were monitored by their DiR fluorescence intensities (ex= 710 nm; em= 800
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nm) in tumors/organs (for biodistribution studies) and in blood (for pharmacokinetic analysis) by
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IVIS imaging at various time points. For the spherical nanoparticles, circulation half-lives
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decreased as a function of increasing particle size (Table 1 and Supplementary Figures S4-
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S6), which was consistent with previously reported studies47. The worm-like micelles (100 nm in
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length) had a t1/2 = (6.6 + 1.9) h (Figure 4A and Supplementary Figure S7), which was
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significantly shorter than the t1/2 of the 100 nm-diameter spherical nanoparticles (10.9 + 1.0 h;
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Table 1). Fluorescence imaging (of nanoparticles) was spatially mapped to luminescence signals
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(from the tumors; e.g., as depicted for the 100 nm-length worm-like micelles in Figure 4A) to
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determine the extent of nanoparticle accumulation within tumors as compared to other organs.
(LUC;
for
in
vivo
imaging)
and
green
fluorescence
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protein
(GFP;
for
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Note that 80-90% of the maximal tumor accumulation for each nanoparticle formulation was
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achieved by 24 h after intravenous administration (Supplementary Figure S4-S7).
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The 300-nm diameter spherical nanoparticles displayed minimal accumulation in subcutaneous
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(SQ) tumors as compared to their 100 nm- or 70 nm-diameter spherical counterparts or to the
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100 nm-length worm-like micelles (Supplementary Figure S4-S7); in particular, the later
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construct showed improved tumor accumulation (Table 1), supporting its further development as
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a pancreatic drug delivery platform. At 72 h post-injection, mice were sacrificed and their major
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organs were dissected to measure DiR fluorescence distribution ex vivo (e.g., Figure 4A, right
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insert). The fluorescence radiant efficiency of every organ was normalized to that of the liver in
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the same mouse (Figure 4B). For all injected suspensions (except for the 300 nm-diameter
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nanospheres), the tumors showed the highest accumulation of PEO-b-PCL-based nanoparticles
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as compared to all other organs (n=5 mice per formulation).
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There were no significant differences in the relative tissue distributions of 70 nm- and 100 nm-
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diameter spherical nanoparticles as compared to the 100 nm-length worm-like micelles, revealing
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similar ex vivo tumor distribution profiles for the injected doses. In contrast, the relative
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accumulation of the 300 nm spherical nanoparticles was lower in the tumors than in the liver and
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was highest in the spleen, consistent with its clearance by the mononuclear phagocytic system
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(MPS)47. Together, these data suggest that 100 nm-diameter nanoparticles may be delivered
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efficiently to allograft PDAC tumors in vivo. While exhibiting similar relative ex vivo biodistribution
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profiles (at 24 h after injection), the worm-like micelles demonstrated a shorter overall circulation
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half-life and an enhanced in vivo tumor accumulation as compared to the spherical nanoparticles
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(Table 1), which possessed comparable overall sizes and which were constructed from the same
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PEO-b-PCL composition, highlighting the potential for shape alterations to improve nanoparticle
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Delivery of PEO-b-PCL-based nanoparticles to autochthonous PDAC tumors in mice
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Previous work has suggested significant differences in vascularity, blood flow, and
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chemotherapeutic drug delivery between transplanted and autochthonous models of PDAC8. In
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particular, autochthonous mouse models that harbor mutations in the proto-oncogene Kras and
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the tumor suppressor gene p53 generate pancreatic tumors with a dense desmoplastic stroma33,
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34, 43;
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and, they exhibit hypoperfusion and reduced drug delivery as compared to allograft models8. As
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discussed previously (vide supra), the potential for vascularity to affect nanoparticle delivery to
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pancreatic tumor cells has been explored using xenograft models of human cell lines that induce
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extensive stroma, suggesting a strong dependence on nanoparticle size. No prior work has
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examined the effects of size on nanoparticle accumulation in autochthonous Kras;p53 mutant
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tumors or those of nanoparticle shape on ultimate drug delivery to hypoperfused pancreatic
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tumors.
they demonstrate the hallmark genetic and histologic features observed in human tumors42;
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We generated Kras;p53 mutant tumors, using a genetically-engineered immunocompetent mouse
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model of PDAC,8, 32, 33 and administered our PEO-b-PCL-based nanoparticles (of varying size and
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shape) via IV tail-vein injection. Whole-animal fluorescence imaging by IVIS was used to
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independently monitor tumor growth (by tomato red fluorescent protein - RFP; see Materials and
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Methods) and nanoparticle location (by DiR; Figure 4C). 24 h after nanoparticle injection, mice
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were sacrificed and their organs were harvested to measure the relative ex vivo fluorescence
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distribution of the tumor cells and the nanoparticles. The harvested pancreas revealed strong
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RFP (Figure 4D) and DiR signals (Figure 4E), verifying successful delivery of the polymeric
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nanoparticles to the primary pancreatic tumor(s) in the autochthonous model. Note that
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normalization of emission intensities where conducted with respect to the organ that displayed
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the largest value in each data set, which was the pancreas in the case of RFP emission (i.e., the 11 ACS Paragon Plus Environment
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organ of highest PDAC cell localization) and the liver in the case of DiR emission (i.e., the healthy
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organ of greatest NP accumulation). A high RFP signal was also detected in the dissected
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intestines and stomach, likely due to the local spread of primary tumor cells. While the 100-nm
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diameter nanospheres appeared to demonstrate higher tumor accumulation than the more
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modest accrual levels displayed by the 300-nm diameter nanospheres, the results obtained with
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either formulation were not significantly different when compared to the relative tumor
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accumulation levels for the 70 nm-diameter nanospheres and the 100 nm-length worm-like
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micelles. These data, thus, reveal that nanoparticles as large as 100 nm are capable of readily
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accumulating in the poorly-permeable pancreatic tumor(s) of the autochthonous model.
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Moreover, we did not observe a significant advantage of the worm-like micelle shape in improving
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delivery to these primary pancreatic tumors, differing from reported studies claiming their
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improved capabilities for solid tumor penetration41.
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To more carefully assess nanoparticle delivery at the cellular level, we analyzed the spatial
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distribution of DiR fluorescence (which identified nanoparticle locations; white) relative to the
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tumor cells (seen by RFP imaging; red) in frozen sections of tumors that were excised from treated
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mice and fixed at 24 h after nanoparticle administration (Figure 4F and Supplementary Figure
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S8). There were no significant differences between the four nanoparticle formulations with respect
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to the mean distances between nanoparticles and their nearest tumor cells (Figure 4G). While
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the overall percentage of the delivered dose was lower, even the 300 nm-diameter nanospheres
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displayed comparable nanoparticle-to-tumor distances as compared to the smaller-sized
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particles. While the possibility exists that differences in tumor accumulation might be observed at
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longer or shorter time points after the administration of different nanoparticle formulations, the
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results would support that once nanoparticles extravasate within pancreatic tumors, their ultimate
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penetration distance is independent of their size or shape.
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Generation of oxaliplatin-loaded nanoparticles
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We next evaluated the capacity of our nanoparticles to deliver a chemotherapeutic agent to
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pancreatic tumors. As a proof-of-principle example, we chose oxaliplatin, which is a platinum-
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containing anticancer agent used in first-line therapy of human PDAC3. To generate an oxaliplatin-
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loaded nanoparticle, we constructed a novel lipophilic oxaliplatin(IV) prodrug (C 16-oxaliplatin(IV)-
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C16), where oxaliplatin was oxidized and conjugated to palmitic acid (Figure 1C). The lipophilic
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prodrug was subsequently encapsulated in the hydrophobic core of PEO-b-PCL-based
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nanoparticles during their fabrication (Figure 1D and Supplementary Scheme S1). By
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measuring the relative fluorescence yield and platinum (Pt) content of the final nanoparticle
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suspensions, we determined that the optimal polymer-to-oxaliplatin (P:O) weight ratio for
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formation was 100:10, suggesting that 10 wt% loading of nanoparticles with the lipophilic
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oxaliplatin(IV) prodrug was the most efficient level for incorporation (Figure 5A). DLS
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measurements revealed that nanoparticles formed at a P:O=100:10 remained ~100 nm in
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dimension (Figure 5B), which was similar to the results obtained with their unloaded PEO5k-
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PCL16k-based counterparts. Cryo-TEM images showed that the DiR-containing PEO-b-PCL-
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based nanoparticles retained a worm-like architecture after oxaliplatin(IV) loading (Figure 5C).
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Thus, it was determined that the oxaliplatin species could be readily incorporated within the
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micelles without altering nanoparticle size or shape.
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We next investigated the drug release profiles of the oxaliplatin(IV)-loaded worm-like micelles.
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PEO-b-PCL-based nanoparticles are known to be stable at neutral pH, while acidic conditions,
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such as those found within endosomes, facilitate release of their contents36, 38. Under neutral or
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acidic pH conditions and in the presence of low concentrations of exogenously added glutathione
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(GSH), platinum (Pt) release remained minimal; at an acidic pH and with higher GSH
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concentrations, which mimicked conditions known to exist within tumor cells
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concentrations of free Pt species were detected in nanoparticle suspensions over time (Figure 13 ACS Paragon Plus Environment
48,
increasing
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5D). Pt release from worm-like micelles was, thus, found to be triggered under acidic and
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reductive in situ conditions, supporting that similar release features could be expected after the
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rapid uptake of the worm-like micelles within in vivo tumor locations. It should be noted that the
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shortened circulatory half-life of oxaliplatin(IV)-loaded worm-like micelles would be expected to
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reduce premature drug release prior to systemic nanoparticle clearance. Given these desirable
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features, we next examined the in vitro potency of the drug-laden micelles as compared to free
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oxaliplatin in both murine (B22) and human (8988T) PDAC cell lines (Supplementary Figure
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S9). From their IC50 values, it was evident that free oxaliplatin and oxaliplatin(IV)-loaded worm-
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like micelles exhibited similar in vitro antitumor activity; note that cellular incubation with unloaded
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PEO-b-PCL-based nanoparticles of varying size and shape did not result in comparable cellular
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toxicity, supporting the biocompatibility of the nanoparticle carrier. Empty nanoparticles that were
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incubated with B22 and 8999T cells at concentrations that were equivalent to the in vivo dose
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used for IV tail-vein injection (i.e., NP1: 5.2 mM or 78 ug/uL of PEO-b-PCL copolymer) did not
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result in significant in vitro cytotoxicity (Supplementary Figures S9D and S9H, respectively).
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In vivo delivery and tumor retention of oxaliplatin(IV)-loaded PEO-b-PCL-based micelles
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Following administration to nude mice harboring B22 subcutaneous pancreatic tumors, we
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compared the delivery and retention of our oxaliplatin(IV)-loaded worm-like micelles with
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equivalent doses of the free drug formulation of unmodified oxaliplatin. Pharmacokinetic analyses
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of the Pt content of blood samples taken at various time points after IV tail-vein injection of free
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oxaliplatin revealed its short circulatory half-life (t1/2 = 1.88 h; Supplementary Figure S10A). The
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circulatory half-life of oxaliplatin was increased by over three-fold (to 6.5 + 0.7 h) when loaded
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within and delivered by DiR-containing worm-like micelles (as measured by DiR fluorescence;
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Supplementary Figure S10B). Note that the half-life of the drug-laden worm-like micelles was
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similar to that of their unloaded counterparts (t1/2 = 6.6 + 1.9 h), suggesting that loading of the Pt
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species does not slow nor accelerate the circulation time of the nanoparticles in blood. 14 ACS Paragon Plus Environment
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We further examined whether the worm-like micelles could enhance drug delivery to poorly
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vascularized tumors, using the autochthonous KPC model of PDAC. The Pt content of various
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organs was determined after IV tail-vein injection of either oxaliplatin(IV)-loaded worm-like
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micelles (NP-oxaliplatin) or free oxaliplatin. NP-oxaliplatin displayed both a relative (with respect
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to other organs) and an absolute increase (~7-fold higher amounts) in the levels of Pt
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accumulation within the pancreas as compared to the free drug formulation at 24 h and as
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assessed by atomic absorption spectroscopy (AAS) (Figure 5E). Note that the pancreas is the
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major organ wherein the tumors are located in these mice; hence, it serves as a surrogate to
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evaluate the extent of intratumoral accumulation. We also measured the amounts of Pt that
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persisted within the pancreatic tumors (as measured by AAS) of SQ-allografted mice that were
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administered either NP-oxaliplatin or free oxaliplatin at identical dose equivalents and that were
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then sacked at similar time intervals. Importantly, the Pt content of the tumors was prolonged after
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worm-like micelle delivery, exhibiting a retention half-life that was ~50x that of free oxaliplatin,
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which was otherwise rapidly cleared from the tumor microenvironment (t1/2 = 5.9 h; Figure 5F).
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Together, these data reveal that the 100 nm-length, PEO-b-PCL-based worm-like micelles are
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capable of delivering drugs to poorly-permeable pancreatic tumors with improved biodistribution
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and prolonged tumor retention as compared to free drug formulations.
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As nab-paclitaxel is an established first-line therapy, and given the recent approval of an
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irinotecan-containing
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considerable interest in pancreatic cancer. To date, however, there have been few published
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studies that have sought to examine the effects of nanoparticle size on tumor penetration in
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murine models of PDAC. Using SQ models, Cabral et al.28 and Wang et al.31 have reported that
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nanoparticles that are sub-30 nm in diameter augment tumor uptake while those that are larger
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than 100 nm exhibit little to no accumulation, presumably due to size-dependent limitations to
nano-liposome17,
novel
nanoparticle
15 ACS Paragon Plus Environment
therapeutics
are
garnering
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nanoparticle extravasation. In contrast to these findings and by using a similar SQ model of
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PDAC, Kobes et al. demonstrated significant tumor uptake for polymeric nanoparticles that were
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greater than 450 nm in size49. Additional investigations have shown that ultra-small nanoparticles
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(< 10 nm in size) demonstrate improved tumor uptake in similar models15, 50.
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A few studies have been performed in more advanced model systems, including in patient-derived
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xenograft (PDX) and in syngeneic orthotopic transplant models of PDAC; they have demonstrated
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enhanced tumor uptake of inorganic nanoparticles that range in size from 40 to 125 nm51, 52. These
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findings, however, contradict those of another study that has compared the sizes of gold
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nanoparticles in a different syngeneic transplant model, determining that nanoparticles that are
