Thermosensitive Liposomal Codelivery of HSAâPaclitaxel and HSA

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Thermosensitive Liposomal Co-delivery of HSApaclitaxel and HSA-ellagic Acid Complexes for Enhanced Drug Perfusion and Efficacy Against Pancreatic Cancer Yan Wei, Yuxi Wang, Dengning Xia, Shiyan Guo, Feng Wang, Xinxin Zhang, and Yong Gan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07132 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 17, 2017

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ACS Applied Materials & Interfaces

Thermosensitive Liposomal Co-delivery of HSA-paclitaxel and HSA-ellagic Acid Complexes for Enhanced Drug Perfusion and Efficacy Against Pancreatic Cancer

Yan Wei,b,1 Yuxi Wang,a,b,1 Dengning Xia,b Shiyan Guo,b Feng Wang,c Xinxin Zhang,b Yong Ganb,*

a

Nano Science and Technology Institute, University of Science and Technology of China,

166 Renai Road, Suzhou, Jiangsu 215123, China b

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road,

Shanghai 201203, China c

Shanghai Institute of Pharmaceutical Industry, 285 Gebaini Road, Shanghai 201203,

China *

Corresponding author. E-mail addresses: [email protected] (Yong Gan).

Corresponding author. Tel.: +86 21 20231000 1424; fax: +86 21 20231000 1425. 1

These authors contribute equally to this work.

1

ACS Paragon Plus Environment


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ABSTRACT Fibrotic stroma and tumor-promoting pancreatic stellate cells (PSCs), critical characters in the pancreatic ductal adenocarcinoma (PDA) microenvironment, promote a tumor-facilitating environment that simultaneously prevents drug penetration into tumor foci and stimulates tumor growth. Nab-PTX, a human serum albumin (HSA) nanoparticle of paclitaxel (PTX), indicates enhanced matrix penetration in PDA probably due to its small size in vivo and high affinity of HSA with secreted protein acidic and rich in cysteine (SPARC), overexpressed in the PDA stroma. However, this HSA nanoparticle shows poor drug blood retention because of its weak colloidal stability in vivo, thus resulting in insufficient drug accumulation within tumor. Encapsulating HSA nanoparticles into the internal aqueous phase of ordinary liposomes improves their blood retention and the following tumor accumulation, but the large 200-nm size and shielding of HSA in the interior might make it difficult for this hybrid nanomedicine to penetrate the fibrotic PDA matrix and promote bioavailability of the payload. In our current work, we prepared ~9-nm HSA complexes with an antitumor drug (PTX) and an anti-PSC drug (ellagic acid, EA), and these two HSA-drug complexes were further co-encapsulated into thermosensitive liposomes (TSLs). This nanomedicine was named TSL/HSA-PE. The use of TSL/HSA-PE could improve drug blood retention, and upon reaching locally heated tumors, these TSLs can rapidly release their payloads (HSA-drug complexes) to facilitate their further tumor accumulation and matrix penetration. With superior tumor accumulation, impressive matrix penetration, and simultaneous action upon tumor cells and PSCs to disrupt PSCs-PDA interaction, TSL/HSA-PE treatment combined with heat exhibited strong tumor growth inhibition and apoptosis in vivo. KEYWORDS:

Pancreatic

cancer,

fibrotic

matrix,

pancreatic

stellate

cells,

thermosensitive liposomes, co-delivery, HSA-drug complexes, PSCs-PDA interaction

2


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1. INTRODUCTION As one of the most malignant human tumors, pancreatic ductal adenocarcinoma (PDA) is characterized by the highest amount of stroma compared with other solid tumors, with the tumor volume consisting of only around 10% cancer cells and the remaining 90% of stroma.1 The stroma is composed of cellular elements such as pancreatic stellate cells (PSCs), endothelial and immune cells, and non-cellular matrix components such as collagen, hyaluronic acid, and secreted protein acidic and rich in cysteine (SPARC).2 This rich stroma creates a tumor-facilitating environment that not only prevents drugs from reaching tumor cells but also stimulates tumor growth. Nanomedicines could accumulate in solid tumors through the enhanced permeability and retention (EPR) effect to improve the tumor accumulation of chemotherapeutic agents. However, this dense fibrotic extracellular matrix (ECM), containing poor vasculature and high intratumoral pressure, forms a solid physical barrier to the delivery of nanomedicines to the peritumoral milieu, resulting in poor tumor-targeting and penetrating performance.3,4 Previous studies showed that only 30 nm small nanoparticles could widely penetrate the fibrotic matrix of PDA to achieve an antitumor effect, whereas 70 nm and 100 nm large nanoparticles remain in close proximity to the vasculature and hardly penetrate tumor matrix, thus resulting in no tumor regression.5 In addition, the stromal cells of PSCs promote tumor cells’ proliferation and reduce their apoptosis, thus facilitating tumor cells’ survival and even resistance to chemotherapy.6,7 Obviously, both the solid physical barrier to drug delivery and tumor-promoting PSCs can seriously compromise the effect of chemotherapy on PDA. However, despite many years of research, conventional chemotherapy regimens still are the option for most patients with advanced PDA, thereby ignoring the complex PDA microenvironment and producing only modest survival benefits. Consequently, there is an urgent need to develop new strategies to enhance drug perfusion to the tumor milieu, as well as simultaneously act on PSCs during tumor chemotherapy.

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Many new strategies such as hyaluronidase,8 CD40 agonists,9 and hedgehog pathway inhibitors10, etc. have been utilized to improve matrix penetration of drugs via targeting different matrix components in PDA. Among these strategies, especially human serum albumin (HSA), a major component of serum proteins, is a natural carrier of hydrophobic molecules with favorable, noncovalent binding characteristics11 and able to improve the tumor matrix penetration of its payload, e.g. paclitaxel (PTX). nab-PTX, a PTX-loaded HSA nanoparticle, is a successful example of an HSA delivery system that is developed based on noncovalent binding of PTX with HSA. Upon injection into the blood, these large 130-nm nab-PTX particles rapidly dissolve into smaller complexes (7 nm on average) consisting of individual HSA-PTX dimers.12,13 Based on 7-nm small particle size in vivo and specific binding with the overexpressed extracellular matrix protein of SPARC in PDA,14-17 these complexes show good stromal penetration and addition of nab-PTX to standard gemcitabine has increased the survival of patients with PDA.18 Therefore, nab-PTX was approved by the FDA in 2013 for the treatment of PDA. However, this nanomedicine has provided only modest survival benefits, probably due to its insufficient tumor distribution.19 Rapid dissociation of nab-PTX results in its poor drug blood retention, with no improvement compared with PTX solution (Taxol) and less retention than other PTX-loaded nanoparticles.20,21 Generally, the stronger blood retention of the nanoparticles remains in circulation, the higher likelihood they have of entering the tumor. Therefore, the poor blood retention of Nab-PTX probably limits its tumor distribution. Recently, a strategy to encapsulate HSA nanoparticles into the internal aqueous phase of ordinary liposomes has significantly improved the plasma half-life and following tumor accumulation of PTX in melanoma compared with that of HSA-PTX nanoparticles.22 But the 200-nm large size of liposomes and shielding of HSA in the interior might make it difficult for these hybrid nanoparticles to penetrate deeply into poorly permeable PDA to reach tumor foci and subsequently exert therapeutic effects.5,23 Therefore, simple liposomal encapsulation of HSA-PTX nanoparticles greatly helps 4


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enhance drug blood retention and following tumor accumulation, but could not simultaneously achieve good matrix penetration in PDA. Preferably, those nanocarriers, coupling

a

specific

stimuli-responsive

drug

release

in

response

to

tumor

microenvironmental stimuli (e.g., heat, pH, or enzyme), would probably be fit to handle this situation because they could suppress drug release while circulating in the bloodstream and actively release their payload after delivery to the tumor tissue. Many enzyme-,24-26 pH-,27,28 and heat-responsive29 nanocarriers have been utilized to improve and optimize drug distribution within tumor. Especially thermosensitive liposomes (TSLs), as heat-responsive nanocarriers, could retain the encapsulated drugs in the bloodstream at 37 oC to enhance drug blood retention, and rapidly release drugs to facilitate their following bioavailability upon reaching locally heated (42-45 oC) tumors. Therefore, we think that TSLs, instead of ordinary liposomes, used for encapsulation of nab-PTX might simultaneously achieve better drug blood retention, tumor accumulation and matrix penetration in PDA. In this study, based on an in-depth understanding of the fibrotic stromal barrier to drug delivery and the tumor-promoting effect of PSCs in PDA, and characteristics of HSA nanoparticles, we developed a novel nanomedicine with TSLs encapsulating HSA complexes of both anti-PSC and antitumor drugs to improve the tumor distribution and subsequent matrix penetration of both drugs for PDA therapy (Scheme 1). Ellagic acid (EA), which has a high affinity for HSA,30-33 inhibits PSC proliferation.34,35 HSA-PTX and HSA-EA complexes were prepared using a self-assembly method via noncovalent binding of HSA and drugs. Then, both these complexes were further encapsulated into TSLs using a thin-film hydration method. This nanomedicine was named TSL/HSA-PE (TSLs integrating HSA-PTX and HSA-EA complexes). Firstly, TSLs could retain both HSA-drug complexes in the blood circulation, thus improving both drugs’ blood retention. Higher drug concentration and longer half-time in blood will reasonably lead to more drug accumulation within tumor. Secondly, after reaching heated tumor, TSL/HSA-PE 5



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could rapidly release both HSA-drug complexes in response to heat stimuli. Based on high affinity of HSA with SPARC and smaller particle size, HSA-PTX and HSA-EA complexes will be able to deeply penetrate tumor matrix, and reach in proximity to and be actively absorbed by tumor cells and PSCs to take effect, respectively. In this study, an avascular BxPC-3&HPaSteC tumor spheroid model was established to evaluate in vitro tumor penetration and growth inhibitory effect of TSL/HSA-PE. The plasma pharmacokinetic (PK) behaviors of PTX were measured to investigate the blood retention effect of TSL/HSA-PE. Fluorescence imaging of in vivo real-time and frozen tumor slices, and determination of drug uptake within tumor were conducted to qualitatively and quantitatively analyze the in vivo tumor-targeting efficiency of TSL/HSA-PE. Finally, the in vivo therapeutic efficacy of TSL/HSA-PE was evaluated using a BxPC-3&HPaSteC-bearing nude mouse model.

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Scheme 1. Schematic illustration of (A) the preparation method of the novel nanomedicine TSL/HSA-PE as well as (B) its application for in vivo therapy against PDA. 2. EXPERIMENTAL SECTION 2.1. Materials. Human serum albumin (HSA) was purchased from Shanghai Xinxing Pharmaceutical co., LTD (Shanghai, China). PTX was obtained from Dalian Meilun Biotech Co., Ltd (Dalian, China). EA was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Dipalmitoylphosphatidylcholine (DPPC) was purchased from Shanghai Advanced Vehicle Technology Pharmaceutical L.T.D. Co (AVT). Fluorescein isothiocyanate (FITC), Brij78, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and 4',6-diamidino-2-phenylindole (DAPI) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Cy7 mono-reactive NHS Ester was purchased from Beijing Fluorescence Biotechnology Co. Ltd (Beijing, China). Tissue-Tek O.C.TTM Compound was ordered from Sakura Finetek (USA). Sephadex G-50 and agarose gel CL-4B were purchased from Shanghai Jianglai Biological Technology Co., Ltd (Shanghai, China). Anti-α-smooth muscle actin (α-SMA) rabbit polyclonal primary antibody was obtained from abcam (UK), and CD31 goat polyclonal primary antibody was from R&D (USA). Alexa fluor® 594-conjugated donkey anti-rabbit secondary antibody was obtained from thermo (USA), and rhodamine affinipure rabbit anti-goat secondary antibody was ordered from Jackson (USA). RPMI 1640 medium, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and trypsin-EDTA solution (0.25%, trypsin with 0.53 mM EDTA) were purchased from Life Technologies Co. (Grand Island, NY, USA). All other chemicals and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) if not mentioned otherwise. 2.2. Cell lines and animals. Human pancreatic stellate cell line HPaSteC was obtained from ScienCell Research Laboratories and cultured in DMEM containing 10% FBS, 100 7



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U/mL penicillin, and 100 µg/mL streptomycin. The human pancreatic cancer cell line BxPC-3 was obtained from the Cell Bank of the Chinese Academy of Sciences and maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Both cell lines were cultured at 37 °C in a 95% humidified atmosphere containing 5% CO2. Male Sprague Dawley (SD) rats (200 ± 20 g) and male BALB/c nude mice (20 ± 2 g) were ordered from Shanghai Sippr-BK Laboratory Animal Co. Ltd. All animal experiments were conducted under the guidelines approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. 2.3. Preparation of TSL/HSA-PE. HSA-PTX complexes were prepared by a previously described self-assembly method with minor modifications.36 An aqueous solution of HSA (2 mg/mL) was incubated with PTX (pre-dissolved in ethanol) at an HSA:drug molar ratio of 1:5 and stirred for 3 hours. Then, the complexes were centrifuged at 8000 rpm for 10 min to remove unloaded drug precipitates, and ethanol was removed through ultrafiltration (MWCO = 10 kDa). HSA-EA complexes were prepared using a similar procedure except EA was pre-dissolved in NMP and added at an HSA:drug molar ratio of 1:10. TSL/HSA-PE was prepared with a thin-film hydration method. Briefly, DPPC and Brij78 were dissolved in chloroform and methanol (9:1, v/v) in a molar ratio of 96:4, followed by the removal of organic solvents using a rotary evaporator under vacuum until a homogeneous lipid film was formed. The lipid film was hydrated with a solution containing HSA-PTX and HSA-EA complexes at PTX and EA feeding concentration of 0.5 mg/mL at 60 °C for 30 min. The suspension was extruded 5 times through 200-nm polycarbonate membranes using an Avanti mini Extruder at 60 °C. Unloaded HSA-drug complexes were removed by gel filtration through sepharose CL-4B. Blank TSL, TSL loaded with HSA-PTX complexes (TSL/HSA-PTX), TSL loaded with HSA-EA complexes (TSL/HSA-EA), and TSL loaded with HSA (TSL/HSA) were prepared using 8


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the same method. All these formulations were stored at 4 °C until use. 2.4. Characterization of TSL/HSA-PE. HSA-PTX, HSA-EA, and TSL/HSA-PE were suitably diluted in 0.01 M PBS, and their size and zeta potential (ZP) were determined using dynamic light scattering (DLS) at 25 oC in a Nano ZS zetasizer (Malvin, UK). The morphologies of different formulations were observed using a cryo-TEM (Tecnai 12 electron microscope, USA) at an accelerating voltage of 200 KV. The phase transition temperature (Tm) of this TSL was determined using DSC (for details, see Supporting Information). PTX or EA was released from TSL/HSA-PE using methanol and concentration was determined through high performance liquid chromatography (HPLC) (for details, see Supporting Information). Drug-loading capacity (DLC) was calculated as indicated in Eq. (1). The DLC values were reported as mean ± SEM (n = 3). DLC (%) =

weight of the drug × 100% weight of the carrier and drug

(1)

For the in vitro temperature-dependent release assay, one volume of TSL/HSA or TSL/HSA-PE in PBS was added to four volumes of pre-heated FBS and incubated at the desired temperatures in a water bath. Following incubation for the designated time, 100 µL of the suspension was pipetted to determine HSA release via increases of fluorescence signals;37 for TSL/HSA-PE another 500 µL of the suspension was removed to determine drug release through ultracentrifugation. FITC-conjugated HSA (FITC-HSA) was synthesized (for details, see Supporting Information) and used to trace HSA release by monitoring increases in fluorescence signals using a microplate reader (Synergy H1, Biotek, USA) at excitation and emission wavelengths of 493 and 525 nm, respectively. Percentage release of HSA was calculated by assuming 100% release with 10 µL Triton X-100 (10%, v/v) and 0% release at 25 oC using Eq. (2): Percentage release (%) =

It - I0 × 100% I max - I 0

(2)

where I0, It, and Imax were defined as signal intensity recorded at 25 oC, at time t, and after adding Triton X-100, respectively. To detect drug release, another 500 µL of the 9



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suspension was diluted using PBS and centrifuged at 120,000 × g for 2 hours at 4 oC to separate released HSA-drug complexes (in the supernatant) from the TSLs (in the precipitates). The released drugs in the supernatant was extracted with methanol, and concentration was determined via HPLC as mt. The suspension at 25 oC, after diluted using PBS and without centrifugation, was extracted with methanol and analyzed as the total amount of mmax. Percentage release of drugs was calculated using Eq. (3): Percentage release (%) =

mt m max

× 100%

(3)

2.5. Cellular uptake and in vitro cytotoxicity assay. To evaluate the cellular uptake of this nanomedicine by BxPC-3 and HPaSteC cells, BxPC-3 and HPaSteC cells were seeded at a density of 3 × 104 cells/well in 12-well plates and incubated for 24 h, respectively. Then, BxPC-3 cells were incubated with FITC-labeled HSA-PTX complexes (NT), TSL/HSA-PTX (NT), TSL/HSA-PTX (HT) for 60 min; HPaSteC cells were incubated with FITC-labeled HSA-EA complexes (NT), TSL/HSA-EA (NT), TSL/HSA-EA (HT) for 60 min. “HT” (hyperthermia) in the brackets denoted that the formulation was kept at 42 °C for 30 min followed by cooling on ice to stop further drug leakage before added to the wells for experiments, while “NT” (normothermia) was used as a control without any preheating. The concentration of FITC was adjusted to 100 ng/mL. Afterwards, the cells were washed three times with cold 0.01 M PBS, mounted in Dako fluorescent mounting medium, and then observed under a confocal laser scanning microscope (CLSM) (FV1000, Olympus, Japan). The in vitro cytotoxicity of different PTX formulations against human BxPC-3 cells and cytotoxicity of various EA formulations against HPaSteC cells was investigated using MTT cell viability assays. BxPC-3 cells were seeded in 96-well plates at a density of 3000 cells/well and allowed to attach for 24 h. Subsequently, the growth media were replaced with Taxol (NT), HSA-PTX (NT), TSL/HSA-PTX (NT) and TSL/HSA-PTX (HT) at PTX concentrations of 0.1, 0.5, 1, 5, or 10 µg/mL. The postfixes of “HT” and 10


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“NT” meant different preheating treatment of the formulations as shown above. MTT assays were carried out after incubation for 48 h. In vitro cytotoxicity of various EA formulations was analyzed using the same procedure except HPaSteC cells were seeded at a density of 5000 cells/well in 96-well plates. 2.6. In vitro penetration and growth inhibition of tumor spheroids. Three-dimensional (3D) spheroids consisting of BxPC-3 and HPaSteC cells were generated to simulate an in vivo fibrotic barrier to drug delivery and the PSCs-PDA interaction. These 3D spheroids were cultured using a previously described liquid overlay method with minor modifications (for details, see Supporting Information).38 The spheroids were stained using toluidine blue to validate the distribution of BxPC-3 and HPaSteC cells based on their different basophilic staining (for details, see Supporting Information).39 The tumor spheroids were cultured at 37 oC until reaching a uniform size of ~400 µm. Then, they were incubated with FITC-labeled HSA-drug complexes (NT), TSL/HSA-PE (NT), or TSL/HSA-PE (HT) with FITC concentration adjusted to 400 ng/mL for 1 or 4 hours, respectively. Here, the postfixes of “HT” and “NT” meant different preheating treatment of the formulations before incubation, as shown in section 2.5. Then, the tumor spheroids were washed with PBS three times, fixed with 4% formaldehyde, and the permeation of fluorescent signals within spheroids was observed under CLSM (FV1000, Olympus, Japan). After the tumor spheroid diameters reached ~400 µm, they were incubated with 400 µL culture media containing Taxol (NT), HSA-PTX (NT), HSA-EA (NT), HSA-PTX + HSA-EA (NT), TSL/HSA-PE (NT), or TSL/HSA-PE (HT) at PTX and EA concentrations of 1 and 0.8 µg/mL, respectively. The postfixes of “HT” and “NT” meant different preheating treatment of the formulations before incubation, as shown in section 2.5. Spheroids treated with drug-free culture media were regarded as the blank controls. The maximum diameter (a) and minimum diameter (b) were measured using an inverted fluorescence microscope (DMI 4000B, Leica, Germany) every three days on Day 0, 3, 6, 11



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9 and spheroid volume (V) was calculated according to the following formula: V = (ab2)/2.40 The percent change curve of spheroid volume was plotted to analyze and compare the tumor inhibitory effects of the various formulations. 2.7. Plasma pharmacokinetics study of PTX. Plasma pharmacokinetics (PK) of PTX was studied to evaluate the drug blood retention effect of TSL/HSA-PE. In detail, male SD rats weighing 200 ± 20 g were randomly divided into three groups (n = 3 for each group). Taxol, HSA-PTX, or TSL/HSA-PE were intravenously administered via the tail vein at an equivalent PTX dose of 5 mg/kg.41 Blood samples were drawn from the retinal vein plexus at preset time points of 5, 15, and 30 min, and 1, 2, 4, 8, 12, and 24 h after intravenous administration and spun immediately at 4000 rpm for 10 min. 150 µL of plasma containing norethindrone as an internal standard was extracted with 4 mL tert-butyl methyl ether via vortexing for 5 min. After centrifugation at 8000 rpm for 10 min, the organic phase was transferred into a clean test tube and dried under nitrogen flow. Then, the residue was redissolved in 100 µL mobile phase and analyzed via HPLC (for details, see Supporting Information). 2.8. In vivo real-time imaging. The fluorescent probe Cy7 was conjugated to HSA by reacting its succinimide ester with HSA according to the manufacturer’s instructions and in vivo behaviors of TSL/HSA-PE were traced. A subcutaneous tumor-bearing nude mouse model was established by inoculating 3 × 106 BxPC-3 and 3 × 106 HPaSteC cells in 100 µL PBS into the subcutaneous tissue of the right hind limbs of nude mice. When the tumors were ready for use, three groups of nude mice were intravenously administrated via the tail vein with 200 µL Cy7-labeled HSA-drug complexes (HT), TSL/HSA-PE (NT), or TSL/HSA-PE (HT) (the dose of Cy7 was 1 mg/kg). “HT” in the brackets denoted that tumors of nude mice were preheated in a 43 °C water bath for 15 min before injection and heating was continued at 43 °C for 1 h after injection while “NT” denoted that the nude mice were not subjected to any heat treatment. The water bath temperature was set at 43 °C to make tumor temperature remain at 42 °C. The 12


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fluorescent images were acquired at 1 h post-injection via an In Vivo IVIS spectrum imaging system (PerkinElmer, USA). Then, the nude mice were euthanized and the hearts, livers, spleens, lungs, kidneys, and tumors were harvested for fluorescent imaging. 2.9. In vivo tumor penetration. A nude mouse model bearing BxPC-3&HPaSteC was generated as described in section 2.8. After the tumors were ready, FITC (1 mg/kg) labeled HSA-drug complexes (HT), TSL/HSA-PE (NT), or TSL/HSA-PE (HT) were injected intravenously to three groups of tumor-bearing nude mice (n = 3). The postfixes of “HT” and “NT” meant different heat treatment of nude mice’s tumors as mentioned above in section 2.8. One hour post-injection, the mice were sacrificed, and the tumors were removed and washed extensively with large volumes of cold saline. To prepare tissue slices, the tumors were embedded in Tissue-Tek O.C.T Compound, frozen at -20 oC, and sliced into 10-µm sections (Leica CM1950, Germany). Finally, the slides were fixed with 4% paraformaldehyde, cell nuclei were counterstained with DAPI, and immunofluorescence staining with anti-CD31 antibody was utilized to label tumor vessels. Fluorescence signals were imaged under a CLSM (FV1000, Olympus, Japan) to investigate the tumor penetration of various formulations. 2.10. Tumor distribution of PTX and EA. The subcutaneous BxPC-3&HPaSteC tumor-bearing nude mouse model was established as shown in section 2.8. When tumor weight reached 0.3−0.4 g, the tumor-bearing nude mice were randomly divided into eight treatment groups (n = 3): Taxol (NT or HT), HSA-PTX (NT or HT), HSA-PTX + HSA-EA (NT or HT), or TSL/HSA-PE (NT or HT). All groups were subjected to i.v. at PTX and EA doses of 5 and 4 mg/kg, respectively. The postfixes of “HT” and “NT” meant different heat treatment of nude mice’s tumors as shown in section 2.8. For the same formulation, different heat treatment of HT or NT was applied to validate effect of heat on drug uptake within tumor. The mice were sacrificed and tumor tissues were harvested at 1 h post i.v. The tumor tissues were weighed and homogenized, and PTX and EA accumulation within tumor was measured via HPLC after extraction (for details, see 13



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Supporting Information). 2.11. In vivo antitumor effects. The subcutaneous BxPC-3&HPaSteC tumor-bearing nude mouse model was established as described in Section 2.8. The tumor-bearing nude mice were randomly divided into six groups (n = 6): saline (HT), Taxol (HT), HSA-PTX (HT), HSA-PTX + HSA-EA (HT), or TSL/HSA-PE (HT or NT). These treatments were intravenously injected at doses of PTX 5 mg/kg and EA 4 mg/kg. The postfixes of “HT” and “NT” meant different heat treatment of nude mice’s tumors as shown in section 2.8. When tumor diameters reached 3−5 mm, treatment was initiated and given every three days for a total of four injections. The body weight and tumor longest (a) and shortest diameters (b) of the mice were measured in 3-day intervals. Tumor volume (V) was calculated using the formula V = (a × b2)/2. After the experiment ended, the mice were sacrificed, and tumor xenografts were excised and weighed. Then, they were fixed with 4% paraformaldehyde for 48 h, embedded in paraffin, and sectioned at 3-µm thickness. The slices were routinely stained using hematoxylin and eosin (H&E) to detect tumor cell necrosis and apoptosis, immunofluorescence stained with anti-α-SMA as an activated PSCs marker accompanied with DAPI counterstaining, and further imaged under a CLSM (FV1000, Olympus, Japan). We determined PSC number by counting the cells in three different and representative fields (200 µm × 200 µm) for each sample. 2.12. Statistical analysis Significant differences were evaluated using an independent-samples t test or Wilcoxon rank test, and multiple treatment groups were compared within individual experiments by Analysis of Variance (ANOVA). p values less than 0.05 were considered significant. Data are presented as mean ± Standard Error of Mean (SEM). 3. RESULTS AND DISCUSSION 3.1. Characterization of TSL/HSA-PE. TSLs, composed of DPPC and Brij78, were used to encapsulate HSA-PTX and HSA-EA complexes to improve drug retention in blood and facilitate their rapid release at tumor site. Here, optional encapsulation of 14


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HSA-drug complexes instead of the parent drugs into TSLs was mainly based on the following facts. Firstly, incorporation of the hydrophobic parent drugs (such as PTX) into DPPC bilayer could induce a broadening of TSLs’ phase transition, thus reducing TSLs’ sensitivity to heat and retarding drug release under heat stimuli.42,43 In contrast, encapsulation of HSA-drug complexes within inner aqueous phase of TSLs does not interrupt the heat-triggered phase transition. Secondly, encapsulating HSA-drug complexes was more conducive to drug uptake within tumor. Compared with parent drugs, HSA-PTX complexes tended to go through the endothelial cells into tumor interstitial space via gp60 receptor-mediated transcytosis,44 and be retained there because of their high-molecular-weight and the lack of lymphatic drainage in tumor.45 In addition, HSA could promote matrix penetration46 and cellular uptake47 of drugs in tumor. TSL/HSA-PE was prepared and further characterized with size, ZP and cryo-TEM imaging. Firstly, both complexes were prepared by self-assembly method with a size of ~9 nm by DLS, slightly larger than that of free HSA (~7 nm), indicating noncovalent binding of both drugs to HSA (Figure 1A and Table S1). This was consistent with previous reports that both PTX and EA have high affinity and binding to HSA.30-33,48 The particle size of blank TSL was initially 132.6 nm. After encapsulating these two HSA-drug complexes using a thin-film hydration method, the particle size of TSL/HSA-PE was 176.2 nm (Figure 1A and Table S2). This increase in TSL/HSA-PE particle size was mainly due to encapsulation of HSA-drug complexes. Second, both HSA-drug complexes had a negative ZP of ~-11 mV, but ZP of TSL/HSA-PE was 0.89 mV, close to the ZP of blank TSL (1.69 mV) (Table S1 and S2). Moreover, with the increase of feeding concentration of drugs, the DLC, particle size of TSL/HSA-PE grew gradually while ZP remained almost unchanged (Table S2), indicating that the water-soluble HSA-drug complexes might be predominantly entrapped in the aqueous core of the TSLs through passive encapsulation when the phospholipids spontaneously form “vesicle” structure with lipid bilayer during the preparation process.49 Thirdly, 15



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cryo-TEM imaging revealed that HSA-EA and HSA-PTX had a similar size of < 10 nm, and TSL showed a size of > 100 nm, which was smaller than the size of TSL/HSA-PE (Figure 1B), consistent with the size values determined by DLS. Both blank TSL and TSL/HSA-PE were unilamellar with a fairly spherical shape. Moreover, cryo-TEM images of TSL/HSA-PE showed a uniform density similar to the density of blank TSL, indicating that there were no free drug crystals and HSA-drug complexes existed in an amorphous form in the liposomal internal aqueous phase. Besides low-MW therapeutic agents such as DOX, many TSLs are also able to temperature-dependently release several nanometer-sized high-MW payloads such as cytokines,50 HSA,51 and dextran.37 The release behaviors of TSL/HSA-PE were determined to identify if this nanomedicine could rapidly release its ~9-nm payloads under heat stimuli. Firstly, free HSA indicated a temperature-dependent release profile: as the temperature went up, a higher percentage of HSA was released. Tm of this TSL, determined by DSC, was 41.14 oC (Figure S1). Especially, at 42 oC, higher than Tm of the TSL, a high percentage (76.9%) of HSA was rapidly released at 10 min (Figure 1C), which was comparable to release percentage of HSA above Tm from other TSLs in previous studies. For example, the TSLs, composed of DPPC/DSPC/PGlcUA and DPPC/MSPC/DSPE-PEG2000, released ~50%50 and ~80%51 of HSA under heat stimuli, respectively. Secondly, HSA-drug complexes showed similar release behaviors to free HSA. After incubation for 1 h in 50% FBS (simulating the serum) at 37 oC, there was no significant change in the size and surface charge of TSL/HSA-PE (Figure S2). This was probably because the superficial PEGylation of this nanomedicine prevented TSLs’ interaction with serum proteins, thus inducing less drug leakage (< 20% at 1 h) at 37 oC in circulation (Figure S2 and 1C).52 While at 42 oC, above Tm, ~70% of HSA-drug complexes

were

rapidly

released

at

10

min

in

FBS

(Figure

1C).

The

temperature-dependent release characteristic of the TSL showed that it could retain its payloads in serum at 37 oC and could rapidly release most of its payloads in 42 oC-heated 16


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tumors. Therefore, this TSL formulation was applicable for in vivo delivery of HSA-drug complexes. Generally, lysolipid-containing TSLs rely on nanopores and defects generated in the lipid bilayer upon heating to release the payloads.53 Due to a relatively large head group relative to their single hydrocarbon tail, lysolipids have a positive intrinsic curvature which favors the formation of micelles. As Tm is approached, grain boundaries begin to melt and lateral lipid mobility increases. Then, the lysolipids would accumulate at the boundaries, and the tendency for lysolipids to form highly curved micelles would lead to the formation of stabilized nanopore in the bilayer.53 Moreover, two-tailed DSPE–PEG, with a negative intrinsic curvature due to the small PE headgroups, can help to some extent in the formation and stabilization of the nanopore structure by bringing a repulsive force within the nanopore.53 For example, another lysolipid-containing TSL consisting of DPPC/MSPC/DSPE-PEG2000 generates 10-nm pores in its lipid bilayer when heated to above Tm.53 Hence, we inferred that this lysolipid-containing TSL released the nano-sized HSA molecule and HSA-drug complexes by forming > 9-nm nanopores in lipid bilayer when heated.

17



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Figure 1. (A) Hydrodynamic diameters of HSA-EA, HSA-PTX, TSL, and TSL/HSA-PE measured using DLS. (B) Cryo-TEM images of HSA-EA, HSA-PTX, TSL, and TSL/HSA-PE. (C) Temperature-dependent release profile of (left) free HSA and (right) HSA-drug complexes from the TSL in PBS containing 80% FBS. Data are presented as mean ± SEM (n = 3). ***p < 0.001 among the marked groups. 3.2. Cellular uptake and in vitro cytotoxicity assay. Cellular uptake of this nanomedicine by BxPC-3 and HPaSteC cells was evaluated. FITC labeled HSA-PTX or HSA-EA complexes showed the strongest uptake to target cells, while the liposomal formulations (TSL/HSA-PTX or TSL/HSA-EA) displayed comparable uptake relative to HSA-drug complexes with preheating and the poorest uptake without preheating (Figure 2). The results confirmed that this nanomedicine could release most of its payloads, HSA-drug complexes, after preheating at 42 °C for 30 min, which was consistent with the release curve (Figure 1C). Furthermore, HSA-drug complexes showed significantly 18


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stronger cellular uptake compared with their liposomal formulation without preheating (TSL/HSA-PTX (NT) or TSL/HSA-EA (NT), which could be attributed to two reasons. Firstly, superficial PEGylation weakened liposome-cell interactions and reduced the liposome uptake by cells due to steric hindrance by the highly hydrated polymer chains.54 Secondly, rapidly proliferating cells such as pancreatic tumor cells or PSCs tended to be nutrient-poor, which could actively scavenge extracellular HSA via micropinocytosis, thus facilitating cellular uptake of HSA-drug complexes.47 As shown in Table 1 and 2, HSA-PTX and HSA-EA complexes could strongly inhibit growth of tumor cells and PSCs, respectively: IC50 values of 0.88 µg/mL for HSA-PTX against BxPC-3 cells, and 0.65 µg/mL for HSA-EA against HPaSteC cells. In addition, after preheated liposomal formulations of TSL/HSA-PTX or TSL/HSA-EA showed slightly less or comparably toxic relative to HSA-drug complexes with slightly higher IC50, while liposomal formulations without preheating were significantly less toxic (P < 0.05) than other groups (Table 1 and 2). The higher cytotoxicity of TSL/HSA-PTX (HT) or TSL/HSA-EA (HT) could be attributed to rapid release of HSA-drug complexes after preheating and better cellular uptake of these HSA-drug complexes relative to their counterparts without preheating.

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Figure 2. Cellular uptake of (A) FITC labeled HSA-PTX complexes (NT), TSL/HSA-PTX (NT) and TSL/HSA-PTX (HT) in BxPC-3 cells and (B) FITC labeled HSA-EA complexes (NT), TSL/HSA-EA (NT) and TSL/HSA-EA (HT) in HPaSteC cells after incubation for 60 min. “HT” meant that the formulation was kept at 42 °C for 30 min followed by cooling on ice to stop further drug leakage before added to the wells for experiments, while “NT” was used as a control without any preheating. Scale bar: 50 µm.

Table 1. IC50 values of different PTX formulations for BxPC-3 cells [Data are mean ± SEM. (n = 3)]. IC50 (µg/mL) Taxol (NT)

0.98 ± 0.23*

HSA-PTX (NT)

0.88 ± 0.22*

TSL/HSA-PTX (NT)

2.84 ± 0.81 20


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TSL/HSA-PTX (HT)

1.06 ± 0.15*

*p < 0.05 vs TSL/HSA-PTX (NT).

Table 2. IC50 values of various EA formulations for HPaSteC cells. [Data are mean ± SEM. (n = 3)]. IC50 (µg/mL) EA solution (NT)

0.80 ± 0.21*

HSA-EA (NT)

0.65 ± 0.22*

TSL/HSA-EA (NT)

2.59 ± 0.92

TSL/HSA-EA (HT)

0.75 ± 0.39*

*p < 0.05 vs TSL/HSA-EA (NT). 3.3. In vitro penetration and growth inhibition of tumor spheroids. Liquid overlay method involves maintaining suspension of cancer cells on a non-adherent surface to allow aggregation of cells into spheroids.38 Avascular tumor spheroids prepared by this technology have been widely used to evaluate tumor penetration and growth inhibitory effect of nanoparticles in vitro.55 PSCs are the principal source of fibrosis in the stroma and create a tumor-facilitating environment.56 3D tumor spheroids consisting of PSCs and PDA cells show increased collagenous regions (Collagen is a main fibrotic matrix component.) and decreased perfusion and cytotoxicity of gemcitabine compared with spheroids grown without PSCs.57 Here, avascular 3D tumor spheroids containing BxPC-3 (PDA cells) and HPaSteC cells (PSCs) were constructed to simulate an in vivo tumor fibrotic matrix barrier to drug delivery and the PSCs-PDA interaction. The BxPC-3 and HPaSteC co-cultured tumor spheroids were well-formed, where PSCs (green triangles) showed less basophilic staining than cancer cells (red triangles), and located in the periphery of cancer cells (Figure 3A). The penetration activities of FITC-labeled HSA-drug complexes (NT), TSL/HSA-PE (NT), and TSL/HSA-PE (HT) were monitored by CLSM (Figure 3B). After incubation 21



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with different formulations for 1 h, the spheroids were subjected to CLSM Z-stack scanning and the surface of spheroids was defined as 0 µm. HSA-drug complexes deeply penetrated the spheroids and green fluorescence was clearly observed even at a scanning depth of 50 µm owing to their ~9-nm small size (Figure 3B). For TSL/HSA-PE (NT), the green fluorescence of FITC was mostly located on the periphery of the spheroids at all scanning depths due to its large particle size (176.2 nm) (Figure 3B). Because of the release of HSA-drug complexes with a small size (~9 nm) after preheated, TSL/HSA-PE (HT) showed comparable penetration behavior to HSA-drug complexes at all scanning depths within spheroids (Figure 3B). After 4 hours of incubation, the green fluorescence of HSA-drug complexes and TSL/HSA-PE (HT) was well perfused and widely distributed even at a scanning depth of 90 µm, while the fluorescence of TSL/HSA-PE (NT) was still mostly located on the periphery of spheroids (Figure S3). These results were consistent with previous studies, which showed that nanoparticles with smaller sizes exhibit enhanced penetration within poorly permeable pancreatic tumor spheroids relative to nanoparticles with larger sizes.58 In addition, the good penetration capability of HSA-PTX complexes within the fibrotic stroma of PDA could also be partly attributed to high affinity of HSA with the matrix protein of SPARC.14-17 The tumor spheroids treated with different formulations were imaged on day 0, 3, 6, and 9, and the growth curve of spheroids was profiled (Figure 3C). Tumor spheroids incubated with the treatment groups grew significantly slower than control spheroids (p < 0.001 on day 9). Both HSA-PTX + HSA-EA (NT) and TSL/HSA-PE (HT) groups exhibited the strongest spheroid growth delay and even caused almost complete ablation of spheroids on day 9, which was significantly stronger than the effects of HSA-EA (p < 0.001), Taxol (p < 0.05), HSA-PTX (p < 0.05), and TSL/HSA-PE (NT) (p < 0.05). Combined with the results of the release curve (Figure 1C), penetration experiments within tumor spheroids (Figure 3B), and cellular uptake and cytotoxicity assay (Figure 2 and Table 1,2) described above, the good performance of TSL/HSA-PE (HT), comparable 22


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to that of HSA-PTX + HSA-EA (NT), can be attributed to rapid release of HSA-PTX and HSA-EA complexes after preheated, wide penetration of HSA-PTX and HSA-EA complexes within tumor spheroids, and simultaneous inhibition of PDA cells and PSCs to interrupt PSCs-PDA interaction after cellular uptake.

Figure 3. (A) Toluidine blue staining of BxPC-3&HPaSteC spheroids: HPaSteC cells (green triangles), located in the periphery of cancer cells, with less basophilic staining than BxPC-3 cells (red triangles). Scale bar: 50 µm. (B) Z-stack fluorescence images of BxPC-3&HPaSteC spheroids after incubation with various FITC-labeled drug formulations for 1 h (B-1: TSL/HSA-PE (HT); B-2: TSL/HSA-PE (NT); B-3: HSA-drug complexes (NT)). Scale bar: 100 µm. (C) Left panel contains representative images of BxPC-3&HPaSteC spheroids observed using an inverted fluorescence microscope after treatment with different drug formulations (C-1: Control (NT); C-2: Taxol (NT); C-3: HSA-PTX (NT); C-4: HSA-EA (NT); C-5: HSA-PTX + HSA-EA (NT); C-6: TSL/HSA-PE (NT); C-7: TSL/HSA-PE (HT)). PTX and EA concentrations were 1 and 0.8 µg/mL, respectively. Spheroids without any treatment served as the control. The 23



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corresponding tumor spheroid growth curve is plotted on the right. “HT” meant that the formulation was kept at 42 °C for 30 min followed by cooling on ice to stop further drug leakage before added to the wells for experiments, while “NT” was used as a control without any preheating. Data represent mean ± SEM (n = 5). *p < 0.05, **p < 0.01, and ***p < 0.001 among the marked groups. Scale bar: 200 µm. 3.4. Plasma pharmacokinetics study of PTX. Poor blood retention of nab-PTX is an urgent problem to be solved. And PK behaviors of PTX could typically reflect the blood retention effect of this nanomedicine, because PTX and EA were encapsulated using a similar method and located at the same site of TSL/HSA-PE. Therefore, the pharmacokinetics (PK) of PTX in Taxol, HSA-PTX complexes (in vivo form of nab-PTX), and TSL/HSA-PE formulations was measured in tumor-free SD rats without hyperthermia to identify if liposomal encapsulation could improve the blood retention of the payloads, which is a prerequisite for enhanced drug delivery to tumor. The PK curves of PTX plasma concentration vs time after i.v. were plotted (Figure 4). Taxol concentration showed a continuous sharp drop and went under the detection limit after 4 h post injection. HSA-PTX complexes with lower initial concentration showed a slower concentration decrease than Taxol, and their concentration-time curve became flat and was located above the Taxol curve at 4 h post-administration. Similar PK behaviors of Taxol and nab-PTX were shown in previous studies that Taxol shows higher initial concentration with a sharp decrease while nab-PTX shows lower initial concentration with a flat decrease.20 The plasma PK for TSL/HSA-PE was greatly prolonged over the HSA-PTX complexes, whose PK curve was located above the curve of HSA-PTX complexes at all timepoints. The parameters of AUC (area under the curve), t1/2 (half-life time), CL (clearance), and Vz (the apparent volume of distribution) were calculated using noncompartmental analysis (Table 3). Firstly, TSL/HSA-PE exhibited prolonged PK by increasing the AUC by 1.97-fold (p < 0.001) and 4.72-fold (p < 0.001), reducing CL by 2.06-fold and 24


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5.00-fold although not statistically significant, and improving t1/2 by 4.14-fold (p < 0.01) and 1.46-fold (p < 0.05) relative to Taxol and HSA-PTX complexes, respectively. These results indicated that after encapsulation with TSLs, drug blood retention of HSA-PTX complexes (in vivo form of nab-PTX) was significantly improved. Secondly, for the critical time frame of 1 h after i.v. administration when hyperthermia conditions were implemented in subsequent experiments to trigger drug release, PTX concentration of TSL/HSA-PE was 3.23-fold, 3.79-fold, 4.80-fold, and 5.63-fold at 5, 15, 30, and 60 min, respectively, compared with PTX concentration of HSA-PTX complexes (Figure 4). Therefore, TSL/HSA-PE with higher plasma concentration could lead to more drug uptake within tumor relative to HSA-drug complexes.

Figure 4. PTX concentration-time profiles following i.v. of Taxol, HSA-PTX complexes, or TSL/HSA-PE in rats at PTX dose of 5 mg/kg. Data are presented as mean ± SEM (n = 3).

Table 3. PK parameters of PTX in Taxol, HSA-PTX complexes and TSL/HSA-PE in rats. Data are presented as mean ± SEM (n = 3). Parameters

Taxol

HSA-PTX

TSL/HSA-PE

AUC (µg/L*h)

3561.84 ± 488.12

1484.45 ± 502.22**

7013.93 ± 759.91***, ###

t1/2 (h)

0.80 ± 0.29

2.27 ± 0.44**

3.31 ± 0.63**, #

CLz (L/h/Kg)

1.40 ± 0.20

3.40 ± 1.12

0.68 ± 0.06

Vz (L/kg)

1.61 ± 0.66

11.53 ± 5.53

3.28 ± 0.82

25



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*p < 0.05, **p < 0.01, ***p < 0.001 vs Taxol. #p < 0.05, ##p < 0.01, ###p < 0.001 vs HSA-PTX complexes. 3.5. In vivo real-time imaging. A non-invasive near-infrared fluorescent imaging method was used to measure in vivo tumor targeting efficiency of TSL/HSA-PE in BxPC-3&HPaSteC-bearing nude mice (Figure 5). TSL/HSA-PE (HT) exhibited the highest fluorescence intensity in the tumor, while TSL/HSA-PE (NT) displayed a lower intensity, and HSA-drug complexes (HT) showed the lowest Cy7 signals at tumor foci (Figure 5A). The main organs were harvested 1 h after administration, and significantly higher fluorescent signals were detected in tumors of animals exposed to TSL/HSA-PE (HT) compared with animals exposed to HSA-drug complexes (HT) (p < 0.01) and TSL/HSA-PE (NT) (p < 0.05) (Figure 5B, C). Semi-quantitative analysis indicated that the tumor fluorescence intensity of TSL/HSA-PE (HT) was approximately 207% and 297% of the signal intensity from TSL/HSA-PE (NT) and HSA-drug complexes (HT), respectively (Figure 5C). Firstly, stronger tumor distribution of TSL/HSA-PE (HT) compared with that of TSL/HSA-PE (NT) was probably attributed to its more extravasation from blood vessels into tumor interstitial space. TSL/HSA-PE (NT) mainly existed as ~176-nm nanoparticles without heat treatment, while TSL/HSA-PE (HT) immediately released most of its payloads, ~9-nm HSA-drug complexes, at heated tumor. It was reported that vascular permeability of the transported nanoparticles based on EPR effect of tumor decreases with increased particle sizes, because not all tumor vessels are leaky and pore size distribution is heterogeneous, leading to heterogeneous extravasation and delivery.59-61 And the vasculature of pancreatic tumor is moderately permeable with pore sizes of 50-60 nm.62 Therefore, vascular permeability of ~176-nm TSL/HSA-PE (NT) could be limited by vascular pore size of PDA but that of ~9-nm TSL/HSA-PE (HT) not. Moreover, HSA-drug complexes also could pass through the tumor blood endothelial cell layer into tumor interstitial space to deliver the payloads via gp60 receptor-mediated transcytosis.63 This was probably the reason why TSL/HSA-PE (HT) showed improved 26


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tumor accumulation compared with TSL/HSA-PE (NT) despite the identically improved drug retention in blood. Therefore, heat treatment was essential to improve tumor accumulation of TSL/HSA-PE. Secondly, with identical heat treatment, stronger tumor distribution of TSL/HSA-PE (HT) than that of HSA-drug complexes (HT), was mainly due to its stronger drug blood retention (Figure 4) with quick release of higher concentration of HSA-drug complexes (Figure 1C) in the locally heated tumor.

Figure 5. (A) In vivo imaging of BxPC-3&HPaSteC-bearing nude mice administrated with Cy7-labeled HSA-drug complexes (HT), TSL/HSA-PE (NT), or TSL/HSA-PE (HT) at 1 h after i.v. (tumors are marked with a red circle). (B) Ex vivo imaging of major organs at 1 h (from left to right: heart, liver, spleen, lung, kidney, tumor). The red arrows marked the tumor. (C) Semi-quantitative fluorescence intensities of the excised tumors. *p < 0.05, **p < 0.01 among the marked groups.

27



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3.6. In vivo tumor penetration. After evaluation of the tumor accumulation of TSL/HSA-PE, tumor slices were prepared and observed using a CLSM to further investigate its matrix penetration in the tumor parenchyma, which is important for therapeutic agents to widely reach tumor foci to produce therapeutic effects. HSA-drug complexes (~9 nm) with the weakest fluorescence signals showed wide extravasation from the blood vessels and penetration into the surrounding tumor tissue (Figure 6). Although TSL/HSA-PE (NT) (176 nm) showed fluorescence signals of medium intensity, these nanoparticles were mainly entrapped in the tumor blood vessels or perivascular tumor tissue, which might lead to insufficient drug concentration at the main tumor foci (Figure 6). In contrast, TSL/HSA-PE (HT) (~9 nm) not only displayed the strongest fluorescence signals, but also widely extravasated from the tumor blood vessels and deeply penetrated the tumor parenchyma (Figure 6). The tumor penetration behaviors of these formulations were consistent with previous studies that nanoparticle permeation within poorly permeable pancreatic tumors correlates well with the particle size: larger nanoparticles appear to stay near the vasculature, while smaller nanoparticles rapidly diffuse throughout the tumor matrix.5,23 In addition, high affinity of HSA with stroma-overexpressed SPARC also contribute to deep permeation of HSA-PTX complexes and TSL/HSA-PE (HT).14-17 These results of drug penetration within tumor were consistent with the results of in vitro tumor spheroid penetration (Figure 3B). This evidence strongly supported our previous assumption that TSL/HSA-PE in combination with local tumor heating could not only improve tumor accumulation of HSA-drug complexes but also facilitate their subsequent matrix penetration.

28


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Figure 6. Distribution of FITC-labeled HSA-drug complexes (HT), TSL/HSA-PE (NT), and TSL/HSA-PE (HT) in the tumor slices of BxPC-3&HPaSteC tumor xenografts 1 h after i.v. Merge 1 is the combination of CD31 and FITC signals; Merge 2 is the combination of DAPI, CD31, and FITC signals. The cell nuclei counterstained with DAPI. The tumor vessels labeled with anti-CD31 antibody. Scale bars: 50 µm. 3.7. Tumor distribution of PTX and EA. After qualitatively evaluating tumor accumulation and penetration of TSL/HSA-PE, tumor distribution of PTX and EA was measured to further quantitatively characterize the tumor targeting efficiency of this nanomedicine. The intratumoral distribution of different PTX and EA formulations was shown in Figure 7. Firstly, within the heated tumor PTX uptake for HSA-PTX and TSL/HSA-PE was 1.38 and 4.86 µg/g tissue, respectively, and EA uptake for HSA-PTX + HSA-EA and TSL/HSA-PE was 1.25 and 4.75 µg/g tissue, respectively. The results indicated that TSL/HSA-PE significantly improved PTX and EA uptake within the heated tumor by 3.5-fold and 3.8-fold, respectively, compared with HSA-drug complexes (p < 0.001). The improved drug uptake for TSL/HSA-PE (HT) was mainly attributed to the enhanced drug blood retention compared with HSA-drug complexes (HT) (Figure 4). In addition, TSL/HSA-PE displayed improved drug uptake (PTX or EA) relative to other 29



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groups in unheated tumors mainly because the liposomal encapsulation enhanced drug blood retention. Secondly, HSA-PTX and HSA-PTX + HSA-EA induced similar PTX uptake within heated or unheated tumors, indicating that the combined administration of HSA-EA did not affect the PK behaviors of HSA-PTX. Thirdly, heat treatment showed different effect on drug uptake within tumor for liposomal and non-liposomal formulations. PTX uptake within tumor for TSL/HSA-PE (HT) was 2.7-fold of PTX uptake for TSL/HSA-PE (NT) (p < 0.001), and EA uptake within tumors for TSL/HSA-PE (HT) was 2.9-fold of EA uptake for TSL/HSA-PE (NT) (p < 0.001). The enhanced drug uptake within tumor for TSL/HSA-PE (HT) was attributed to heat-triggered release of HSA-drug complexes, which more tended to pass through blood vessels into tumor parenchyma compared with the complete lipid formulation of TSL/HSA-PE (NT). And the results were consistent with the tumor distribution determined qualitatively by in vivo real-time imaging experiments (Figure 5). However, heat showed no significant effect on drug uptake within tumor for non-liposomal formulations such as Taxol, HSA-PTX, and HSA-PTX + HSA-EA. It was reported that hyperthermia increases the vascular pore cutoff size of tumors to facilitate the accumulation of 100-nm liposomes.64 But small particle sizes of these non-liposomal formulation make them freely pass through leaky tumor blood vessels and their tumor accumulation is not restricted by the vascular cutoff size, and thus no drug uptake was enhanced by heat. Consequently, in the following antitumor efficacy experiment, all formulations were combined with hyperthermia except liposomal formulation of TSL/HSA-PE (NT) as an unheated control.

30


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Figure 7. In vivo tumor distribution of PTX and EA in BxPC-3&HPaSteC-bearing nude mice, which were randomly divided into 8 treatment groups: Taxol (NT or HT), HSA-PTX (NT or HT), HSA-PTX + HSA-EA (NT or HT), and TSL/HSA-PE (NT or HT). PTX and EA were administrated at doses of 5 mg/kg and 4 mg/kg, respectively. Data are presented as mean ± SEM (n = 3). ***p < 0.001 among the marked groups. 3.8. In vivo antitumor effects. To further verify in vivo therapeutic effects of this nanomedicine, we treated BxPC-3&HPaSteC-bearing nude mice with TSL/HSA-PE or various other formulations through i.v. after tumor diameter reached 3−5 mm. As indicated in Figure 8A, Taxol (HT) treatment only slightly inhibited tumor growth relative to saline administration. TSL/HSA-PE (NT), HSA-PTX (HT), and HSA-PTX + HSA-EA (HT) showed (in increasing order) improved, moderate tumor growth inhibition. TSL/HSA-PE (NT), with more drug distribution within tumors (Figure 7), showed weaker tumor growth inhibition than HSA-PTX + HSA-EA (HT) owing to its low matrix penetration (Figure 6), thus resulting in poor drug availability. HSA-PTX + HSA-EA (HT) displayed stronger tumor growth inhibition relative to HSA-PTX (HT), because the additional action of HSA-EA on PSCs disrupted the PSCs-PDA interaction. Especially, TSL/HSA-PE (HT) exhibited superior antitumor effects to all other treatment groups, owing to more drug accumulation within tumors (Figure 7), deeper matrix penetration of both HSA-drug complexes (Figure 6), and simultaneous inhibition of tumor cells and 31



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PSCs to disrupt the PSCs-PDA interaction. These results were consistent with growth inhibitory effects of TSL/HSA-PE (HT) on in vitro tumor spheroids (Figure 3C). At the end of treatment, tumor xenografts were excised, imaged, and analyzed, which was consistent with the tumor growth curve (Figure 8B). Body weight of nude mice was weighed to investigate in vivo systemic toxicity of the different treatments (Figure S4). Only Taxol-treatment showed a slight systemic toxicity with a weight loss of < 5%. While for all the other groups, body weight of nude mice showed a slightly increasing trend relative to initial body weight on day 0. Especially, TSL/HSA-PE (HT) showed a comparable weight gain compared with saline and other treatment groups, indicating that this nanomedicine could be potentially safe for PDA chemotherapy. Cellular apoptosis within tumor was further evaluated using H&E staining (Figure 8C). The Taxol (HT)-treated group exhibited more complete tumor cell shape and fewer apoptotic bodies than other treatment groups. Tumors treated with HSA-PTX + HSA-EA (HT) contained more apoptotic cells compared with tumors treated with TSL/HSA-PE (NT) or HSA-PTX (HT). Also, the TSL/HSA-PE (HT)-treated group displayed the lowest density of live cells and the most apoptotic and necrotic cells compared to the other groups. Consistent with the treatment results, TSL/HSA-PE (HT) showed a favorable antitumor effect. Activated PSCs in tumor slices were stained with α-SMA using immunofluorescence to evaluate cell numbers after treatment (Figure 8D and S5). The average number of PSCs in Taxol (HT)- and HSA-PTX (HT)-treated tumors was 93% and 87% of the PSCs number in saline (HT)-treated tumors, indicating nonsignificant decreases in PSCs number. TSL/HSA-PE (NT), HSA-PTX + HSA-EA (HT), and TSL/HSA-PE (HT) treatment resulted in 68%, 51%, and 23% of the PSC number observed upon saline treatment, indicating that stronger tumor distribution and matrix penetration of EA led to a fewer number of PSCs. These results further supported the idea that TSL/HSA-PE (HT) significantly disrupts the PSCs-PDA interaction via inhibiting PSCs growth. 32


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Figure 8. Nude mice bearing BxPC-3&HPaSteC were treated with the indicated formulations for ~2 weeks. These treatments were intravenously injected at doses of PTX 5 mg/kg and EA 4 mg/kg. The postfixes of “HT” and “NT” meant different heat treatment of nude mice’s tumors as shown in section 2.8. (A) Tumor growth curve during the whole experiment was profiled. Data were presented as mean ± SEM (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 among the marked groups and TSL/HSA-PE (HT) group. (B) Representative tumor xenograft images and tumor weight (B-1: Saline (HT); B-2: Taxol (HT); B-3: TSL/HSA-PE (NT); B-4: HSA-PTX (HT); B-5: HSA-PTX + HSA-EA (HT); B-6: TSL/HSA-PE (HT)). (C) Images of H&E-stained tumor slices excised from subcutaneous tumor-bearing mice (scale bar: 100 µm). The green circles marked the apoptotic and necrotic cells. (D) Staining of activated PSCs using α-SMA (red) immunofluorescence for quantification on Day 15 after treatment with the various formulations were shown (scale bar: 100 µm). 33



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4. CONCLUSION In this study, we constructed a liposome-based nanomedicine, TSL/HSA-PE, to enhance drug blood retention, following tumor accumulation, and matrix penetration of both antitumor and anti-PSC drugs. This formulation was designed to inhibit not only tumor cells but also PSCs to interrupt the PSCs-PDA interaction for PDA therapy. In combination with hyperthermia, TSL/HSA-PE rapidly released its loaded HSA-PTX and HSA-EA complexes, and displayed superior penetration and growth inhibition in in vitro BxPC-3&HPaSteC spheroids, similar to the effects of HSA-drug complexes. In addition, TSL/HSA-PE displayed improved blood retention, and when combined with heat, this formulation improved drug distribution within tumor and following matrix penetration, induced the lowest PSC number, and exerted the strongest tumor growth inhibition and apoptosis in BxPC-3&HPaSteC-bearing nude mice compared with other groups. In summary, TSL/HSA-PE treatment with heat might be a potential drug delivery system for the clinical therapy of PDA.

ASSOCIATED CONTENT Supporting Information HPLC condition used for PTX and EA determination; labeling of HSA using FITC; building of in vitro BxPC-3&HPaSteC tumor spheroid model; toluidine blue staining of the tumor spheroids; determination of Tm of the TSL using DSC; determination of PTX uptake within tumor; determination of EA uptake within tumor; characterization of HSA, HSA-PTX, HSA-EA, TSL, TSL/HSA-PE; Tm of the TSL; particle size and ZP of TSL/HSA-PE in 50% FBS; fluorescence penetration in BxPC-3&HPaSteC tumor spheroids after 4 hour incubation with TSL/HSA-PE (HT), TSL/HSA-PE (NT), or HSA-drug complexes (NT); body weight change of BxPC-3&HPaSteC-bearing nude mice during the treatment; a quantification of activated PSCs in the tumors after the treatment. 34


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AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Author Contributions All the authors have contributions for this work and have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The work was supported by China Postdoctoral Science Foundation (grant No. 2016M600342). We would like to thank the National Center for Protein Science Shanghai for cryo-TEM analysis and we are grateful to Li Danyang and Yuan Qingning for their help of making samples and taking pictures.

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