Nanoplatform Assembled from a CD44-Targeted Prodrug and Smart

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Nanoplatform Assembled from a CD44-Targeted Prodrug and Smart Liposomes for Dual Targeting of Tumor Microenvironment and Cancer Cells Yaqi Lv, Chaoran Xu, Xiangmei Zhao, Chenshi Lin, Xin Yang, Xiaofei Xin, Li Zhang, Chao Qin, Xiaopeng Han, Lei Yang, Wei He, and Lifang Yin ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b08051 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

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Nanoplatform Assembled from a CD44-Targeted Prodrug and Smart Liposomes for Dual Targeting of Tumor Microenvironment and Cancer Cells Yaqi Lv, Chaoran Xu, Xiangmei Zhao, Chenshi Lin, Xin Yang, Xiaofei Xin, Li Zhang, Chao Qin, Xiaopeng Han, Lei Yang, Wei He*, Lifang Yin* Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, P.R. China *Corresponding Authors *E-mail: [email protected] *E-mail: [email protected]

ABSTRACT: The tumor microenvironment (TME) plays a critical role in tumor initiation, progression, invasion and metastasis. Therefore, a therapy that combines chemotherapeutic drugs with a TME modulator could be a promising route for cancer treatment. This paper reports a nanoplatform self-assembled from a hyaluronic acid (HA)-paclitaxel (PTX) (HA-PTX) prodrug and marimastat (MATT)-loaded thermosensitive liposomes (LTSLs) (MATT-LTSLs) for the dual targeting of the TME and cancer cells. Interestingly, the prodrug HA-PTX can self-assemble on both positively and negatively charged liposomes, forming hybrid nanoparticles (HNPs, 100 nm). Triggered by mild hyperthermia, HA-PTX/MATT-LTSLs HNPs rapidly release their payloads into the extracellular environment, and the released HA-PTX quickly enters 4T1 cells through a CD44-HA affinity. The HNPs possess promoted tumor accumulation (1.6-fold), exhibit deep tumor penetration, and 1

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significantly inhibit the tumor growth (10-fold), the metastasis (100%) and angiogenesis (10-fold), etc. Importantly, by targeting the TME and maintaining its integrity via inhibiting the expression and activity of matrix metalloproteinases (MMPs) (> 5-fold), blocking the fibroblast activation through downregulating the TGF-β1 expression (5-fold) and suppressing the degradation of extracellular matrix, the HNPs allow for significant metastasis inhibition. Overall, these findings indicate that a prodrug of an HA-hydrophobic-active compound and liposomes can be self-assembled into a smart nanoplatform for the dual targeting of the TME and tumor cells and efficient combined treatment; additionally, the co-delivery of MATT and HA-PTX with the HNPs is a promising approach for the treatment of metastatic cancer. This study creates opportunities for fabricating multifunctional nanodevices and offers an efficient strategy for disease therapy.

KEYWORDS:

thermosensitive

prodrug,

liposomes,

matrix

metalloproteinases,

tumor

microenvironment, extracellular matrix, metastatic breast cancer

The TME plays a critical role in tumor initiation, progression, metastasis, and resistance to therapy. It is composed of non-cancer cells including endothelial cells, immune cells and fibroblasts, an altered extracellular matrix (ECM), and a vascular and lymphatic network.1 Among these elements, the ECM contains a large number of matrix macromolecules and consequently provides physical scaffolding for embedded cells, and it is also a predominant factor influencing cancer cell migration and drive invasion.2 As a result, changes in the ECM regulate the TME and thus affect cellular processes, including invasion and migration.3,

4

Matrix metalloproteinases (MMPs), which are extracellular 2


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proteinases, can degrade ECM components, such as collagens, gelatins and fibronectin, and cleave relevant proteins.5 Therefore, MMPs play a vital role in the promotion of cancer progression and tumor metastasis, especially due to their ability to alter the microenvironment.6 The high expression of MMPs, especially MMP-2 and MMP-9, has been found in a variety of common tumors, such as colorectal carcinomas, breast cancer, brain cancer, and non-small-cell lung cancer.7-11 Previous reports have also suggested that interfering with MMP function or expression blocks the metastatic cascade and tumor growth.12 Accordingly, it is widely assumed that regulating the expression of MMPs in the TME can suppress tumor growth and inhibit metastatic spread.3, 13, 14 Marimastat (MATT), a broad-spectrum synthetic enzyme inhibitor, can achieve greater than 50% enzyme inhibition of collagenases, gelatinases, and MMPs, even at a nanomolar concentration; it mimics MMP substrates and thus displays competitive and potent, but reversible, suppression.12, 15, 16 MATT inhibits tumor progression by inhibiting metastasis. Nevertheless, it is not cytotoxic and therefore cannot kill tumor cells, which dramatically compromises its antitumor efficacy. Consequently, delivering MATT alone to target MMPs in the TME is insufficient to eliminate tumor cells. Thus, a potent approach for cancer therapy based on MATT is highly desirable. Generally, researchers have assumed that overexpressed receptors in cancer cells provide a well-defined strategy for selectively killing tumor cells.17 CD44 is a transmembrane cell-surface protein that is highly expressed on various types of cancer cells, including breast, pancreas, colon, prostate, and stomach cancer,18, 19 and CD44 determines the tumorigenic and metastatic capacities of many cancer cells.20 Hyaluronic acid (HA), a natural acidic polysaccharide that is non-toxic and biodegradable, possesses a strong affinity for CD44 receptors and thus can be utilized as a ligand for 3


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tumor-targeting.21 Paclitaxel (PTX) is one of the most effective cytotoxic drugs for treating diverse solid tumors, especially metastatic breast cancer.22, 23 Treatment with HA-conjugated PTX improves cancer therapy due to the specific binding of HA to CD44.24 Therefore, it is expected that the combination therapy with MATT and an HA-PTX prodrug will have a synergistic effect on the inhibition of tumor growth. However, it has been a great challenge to simultaneously load the prodrug and MATT in a single vehicle for their subsequent delivery and release to target sites. Lysolipid-containing thermosensitive liposomes (LTSLs) are smart nanocarriers that are stable at body temperature (37 °C) but disintegrate during a phase transition that occurs during mild hyperthermia (HT) at 42 °C to promote the rapid release of encapsulated drugs in a local region.25, 26 LTSLs loaded with doxorubicin (ThermoDox®) have entered human clinical trials;27-29 and accordingly, LTSLs are a promising drug delivery system. In this study, hybrid nanoparticles (HNPs) directly self-assembled from an HA-PTX prodrug and MATT-loaded LTSLs (MATT-LTSLs) were developed to deliver MATT and HA-PTX to the TME and cancer cells, respectively, for the treatment of metastatic breast cancer. The formation of this hybrid nanosystem (HA-PTX/MATT-LTSL HNPs) and the proposed mechanism of action after intravenous administration are illustrated in Figure 1. Unlike traditional targeted-delivery routes, this nanosystem was fabricated by assembling a targeting prodrug and another drug-payload liposome that simultaneously accomplishes combined therapy for the disease and active-targeted delivery. Importantly, the versatile HNPs are able to simultaneously target the disease microenvironment and diseased cells, representing an efficient approach of disease therapy. This report indicates that a prodrug composed of insoluble drugs and polymers and liposomes can be assembled directly into a 4


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nano-scaffold. This study provides a strategy for preparing smart nanoparticles for drug delivery. To study the formation of this nanosystem and explore its potential application for disease treatment, comprehensive experiments were performed.

RESULTS Preparation and characterization. HA-PTX/MATT-LTSL HNPs consisted of the prodrug HA-PTX and MATT-LTSLs. HA-PTX was synthesized according to a previous report.30 In brief, HA-PTX was synthesized by adipic dihydrazide (ADH)-modified HA and PTX 2`-OH via a succinate ester bond and was characterized by infrared (IR), ultraviolet and visible spectra (UV/Vis) spectra and 1H NMR (Supplementary Figure S1). The PTX loading rate (%) in the prodrug was approximately 15%, as calculated by the characteristic peak of 1H NMR. MATT-LTSLs were prepared by film hydration followed by the probe supersonic method. The encapsulation percentage of MATT in LTSLs based on high performance liquid chromatography (HPLC) assay was 56.36%. Additionally, cationic temperature-sensitive liposomes (LTDSLs) and long-circulating liposomes (SSLs) were included in the study to study the mechanism of formation for the self-assembled hybrid nanoparticles. Finally, the HNPs were prepared by self-assembling the prodrug HA-PTX with these liposomes at a HA/lipid mass ratio of 1: 4. Table 1 summarizes the main physical-chemical properties of these key nanoparticles. Dynamic light scattering (DLS) showed the MATT-LTSLs and HNPs were 90-100 nm in diameter with a polydispersity index (PDI) less than 0.3. The average particle size and PDI of HA-PTX/MATT-LTSL HNPs with a PTX/MATT mass ratio of 1:1 were a little greater than those of MATT-LTSLs, predominately due to the coating of HA-PTX (Figure 2A). Similar results were obtained with 5


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HA-PTX/LTDSL HNPs and HA-PTX/SSL HNPs. In contrast to MATT-LTSLs with a positive surface charge, a slight negative surface charge (-1.62 mV) was detected in HA-PTX/MATT-LTSL HNPs. Therefore, to some extent, HA-PTX was attached to the surface of MATT-LTSLs. The spherical morphology of MATT-LTSLs and HNPs was confirmed by transmission electron microscopy (TEM) (Figure 2D). Confirmation of coassembly and membrane fluidity measurement. To ascertain the assembly of HA-PTX on liposomes, measurements of fluorescence resonance energy transfer (FRET) and membrane fluidity were performed. First, FRET was utilized to confirm that HA-PTX can be assembled on the liposomes. FRET, a well-defined energy transfer process between a fluorescent donor and an acceptor within 1–10 nm, is an efficient tool to detect the dedicated interplay between nanoparticles and external or internal stimuli by monitoring the changes of fluorescence from both donor and acceptor.31, 32 With this approach, fluorescein isothiocyanate (FITC), a donor, was conjugated with HA-PTX, and rhodamine B (Rho), an acceptor, was encapsulated in LTSLs, LTDSLs, and SSLs, with surface charges of approximately +3 mV, +10 mV and –2 mV, respectively. Strong fluorescence from FITC-HA-PTX (FITC, 515 nm) and low emission from Rho-LTSLs (Rho, 575 nm) were displayed (Figure 2B), which indicated a profound FRET effect from the donor and acceptor. To study the energy transfer, HNPs with various FITC-HA-PTX/Rho-LTSL ratios were prepared, and their fluorescence emission spectra were monitored. Importantly, fluorescence intensity from the acceptor (Rho, 575 nm) increased with an increase of Rho-LTSLs, whereas the donor (FITC) fluorescence at 515 nm gradually declined (Figure 2B).

Similar

results

were

obtained

with

FITC-HA-PTX/Rho-LTDSL

6


HNPs

and

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FITC-HA-PTX/Rho-SSL HNPs (Supplementary Figure S2), which exhibited gradually increased fluorescence quenching of FITC as the Rho in HNPs increased. Inversely, a decreasing trend in the acceptor fluorescence and an increase in donor fluorescence are shown after separating FITC-HA-PTX and Rho-LTSLs from HNPs through a mild hyperthermia process that disintegrates LTSLs (Figure 2C). Figure 2E shows the confocal laser scanning microscope (CLSM) images of 4T1 cells incubated with FITC-HA-PTX/Rho-LTSL HNPs at different ratios. Upon increasing the concentration of Rho-LTSLs (acceptor), red fluorescence intensified while the green fluorescence of FITC-HA-PTX (donor) gradually weakened. Next, we assumed that the membrane fluidity of liposomes would be affected if the HA-PTX assembly was true. Therefore, the measurement of fluorescence anisotropy was performed. 1,6-diphenyl-1,3,5-hexatriene (DPH), a membrane-bound probe that reflects the interference in the hydrophobic region of lipid membrane,33, 34 was loaded in the lipid bilayers of LTSLs in HNPs. As shown in Figure 2F, the anisotropy values obtained from HNPs with different HA/lipid ratios at 25°C or 37 °C was increased by 15%–27% compared with that from LTSLs, demonstrating that the DPH mobility was reduced after the assembly of HA-PTX. The decreased membrane fluidity of LTSLs in turn verified the strong interaction between HA-PTX and LTSLs and confirmed the assembly of HA-PTX on liposomes. Noticeably, at 42 °C closing to the phase transition temperature of LTSLs, the anisotropy values decreased significantly in comparison with that at 25°C or 37 °C. Furthermore, the values from HNPs at 42 °C, despite the HA-PTX/LTSL ratios, were similar to that from LTSLs, indicating the maintenance of thermosensitivity. These data demonstrated that the coating of HA-PTX

7


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reduced the membrane fluidity of LTSLs and that the HNPs possessed similar thermosensitivity with LTSLs. Collectively, the measurements of FRET and fluorescence anisotropy demonstrated that the prodrug HA-PTX can be assembled onto the LTSLs, LTDSLs as well as SSLs. Serum stability and temperature-triggered release. The stability was studied by FRET. After stored in 10% FBS medium for 48 h at 37°C, the FRET ratio in FITC-HA-PTX/Rho-LTSL HNPs was not altered (Figure 2G) and, accordingly, indicated that HNPs were stable against dissociation. To assess the thermosensitivity of prepared LTSLs and determine whether HA-PTX assembled on LTSLs would affect their responses to temperature, an examination of temperature-triggered drug release was conducted for both LTSLs and HNPs. A fluorescence probe, 5(6)-carbocyfluorescein (CF), was loaded into the vehicles to study the release profiles at different temperatures. The assembly of HA-PTX onto the surface of LTSLs had little effect on the release of CF at 37 °C, however, resulted in a retarded release at 42 °C 60 min later (Figure 2H). Nonetheless, at 42 °C, both LTSLs and HNPs released their payloads in a dramatically faster pattern than that at 37 °C (normal body temperature) due to the presence of lysolipids in the lipid bilayer of LTSLs, which leads to a less ordered phospholipid arrangement,31, 35 demonstrating that HNPs had similar thermo-responsive feature as well. This result demonstrated that after an injection into the circulation system, most of the drug would be well encapsulated in HNPs, and rapid drug exposure in a local disease site could be achieved with HT treatment. CD44-targeted intracellular uptake of HA-PTX and trafficking. To study the expression of CD44 receptors on 4T1 cells, CLSM observation and quantification by flow cytometry were performed 8


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after staining with a FITC-labeled CD44 antibody. As depicted in Supplementary Figure S3A, intense green fluorescence on the cell surface was observed, which indicated high expression of CD44 receptors on 4T1 cells. Additional quantitative analysis demonstrated 93.07±1.29% CD44-positive 4T1 cells (Supplementary Figure S3B). For the other intended target, collagen, especially collagen I, and gelatin, which are the two dominant components of the ECM,2 are rich in the 4T1 cell-induced TME and play vital roles in the migration and invasion of tumor cells. Therefore, 4T1 cells were chosen for our cell experiments, and 4T1 tumor-bearing mice were used to study in vivo performance. We first examined whether HNPs would promote a desired pattern using a real-time confocal technique. After reaching the local disease site and undergoing HT treatment at 42 °C, HNPs were disrupted and subsequently released the LTSL-encapsulated payload and HA-PTX, which could rapidly enter cancer cells via CD44 receptor association. To test this approach, hybrid dual-labeled Rho-HA-PTX/CF-LTSL HNPs were prepared by assembling Rho-HA-PTX onto CF-LTSLs. As shown in Supplementary Movie S1 and Figure 3A, after HT treatment, the distribution of green fluorescence from CF in the extracellular medium and profound red fluorescence (Rho) in the cytoplasm were observed and became stronger over time. This result revealed that after delivery to the local disease site and HT treatment, the HNPs were not ready for cellular uptake but released the payloads of CF and HA-PTX into the extracellular environment, and the HA-PTX achieved rapid cellular entry. Next, we investigated the role of the CD44 receptor in the cellular uptake of HA-PTX in 4T1 cells using CLSM and flow cytometry. In contrast with the control (free Rho, Supplementary Figure S4), after culturing the cells, significant fluorescence (red) was visualized in perinuclear regions (Supplementary S5A), and the fluorescence became stronger with increasing Rho concentration and 9


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over time (Figure 3B and Supplementary Figure S5B). We then incubated Rho-HA-PTX with 4T1 cells at different temperatures. The fluorescence intensity of Rho-HA-PTX was significantly higher at 37 °C than at 4 °C (Supplementary Figure S5C, D). These results suggested that HA-PTX was effectively delivered into 4T1 cells in a time-dependent and energy-related manner. To illustrate the contribution of CD44 receptors in cellular uptake, Rho-HA-PTX was incubated with 4T1 cells with CD44 receptors saturated by pre-treatment with an excessive amount of free HA. After incubation with pretreated cells for 4 h with a fixed concentration of Rho-HA-PTX, measurement by flow cytometry showed that the fluorescence intensity was significantly reduced compared to that of untreated cells (Figure. 3C). Visually, CLSM observation also revealed that the red fluorescence (Rho-HA-PTX) distributed in the perinuclear areas of cells pretreated with HA was markedly weaker than that in untreated cells (Figure 3D). Additional quantification indicated that at high concentrations of Rho-HA-PTX, the cellular uptake of Rho-HA-PTX was approximately only 50% of the uptake in untreated cells (Figure 3E). These results confirmed that a CD44-mediated endocytosis pathway driven by CD44-HA affinity played a critical role in the uptake of Rho-HA-PTX. To study the intracellular trafficking of HA-PTX, lysosomes and late endosomes were marked with lyso-tracker green. Yellow spots in the merged images were visualized after the co-localization of red fluorescence (Rho-HA-PTX) and green fluorescence (endo/lysosomes) (Figure 3F), which demonstrated that Rho-HA-PTX was detained by lysosomes before accumulating in cytoplasm. As a result, Rho-HA-PTX obtained cellular entry through an endocytic pathway.36 Moreover, the cellular uptake was significantly reduced by chlorpromazine (Cpz), cytochalasin-D (Cyto-D) and NaN3 with deoxyglucose (NaN3+DG) (Supplementary Figure S6), implying that this uptake was an 10


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energy-dependent process and mediated by micropinocytosis and a clathrin-pathway. In vitro cytotoxicity and synergistic effect. Cell viability in the presence of the drug-free carriers, HA-ADH and LTSLs, was approximately 100%, even at high concentrations (Supplementary Figure S7A, B). Therefore, these carriers are safe. HA-PTX exhibited more profound cytotoxicity against 4T1 cells than free PTX, as the PTX concentration ranged from 1 to 10 μg/mL, mainly due to the improved uptake of HA-PTX via CD44 mediation (Figure 4A). Surprisingly, free MATT appeared to have no cytotoxicity at any tested concentration, whereas MATT-LTSLs displayed significant cytotoxicity at high MATT concentrations independent of HT treatment (Figure 4B). To examine the cytotoxicity of HNPs, we first evaluated the synergistic effects between HA-PTX and MATT by calculating the combination index (CI). As depicted in Supplementary Figure S8 (A, B), at high inhibition rate (Fa), the CI values from the HNPs with PTX/MATT ratios of 3:1, 2:1, 1:1 and 1:2 were less than 1 and therefore demonstrated the synergism between HA-PTX and MATT. However, at MATT concentrations ≥ 5 μg/mL, the ratios of 2:1, 1:1 and 1:2 exhibited higher toxicity against 4T1 cells than the 3:1 ratio (Supplementary Figure S8A), demonstrating promoted in vitro synergistic effect. Indeed, HNPs with PTX/MATT ratio of 1:1 showed greater toxicity to 4T1 cells than MATT-LTSLs or HA-PTX when the concentration of PTX was higher than 1 μg/mL (Figure 4C), which demonstrated synergistic cancer cell killing. Additionally, for HNPs, there was little impact of HT treatment on cytotoxicity at low drug concentrations (< 0.5 μg/mL MATT) (Figure 4C). In vitro cell apoptosis. To observe the apoptosis of cancer cells, the morphology changes of cells treated with these formulations were monitored by fluorescence microscopy (Figure 4D). Changes in the shapes of 4T1 cells occurred for all the drug-containing formulations. However, the most profound 11


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alternations, including chromatin condensation and fragmentation, were seen in the cells incubated with HNPs independent of HT treatment. Further quantitative measurements with flow cytometry indicated that the apoptosis rate of 4T1 cells treated with HNPs +HT was 88% and was significantly higher than that from HA-PTX treatment (Figure 4E). Interestingly, the apoptosis rates upon treatment with MATT-LTSLs and MATT-LTSLs+HT were approximately 3- and 3.6-fold higher, respectively, compared to free MATT. In vitro inhibition of fibroblast activation. Fibroblasts are a kind of non-cancer cells in ECM;37 however, approximately 80% of the fibroblasts in breast cancer stroma would be activated to cancer-associated fibroblasts (CAFs) by various growth factors, cytokines, proteases and hormones such as transforming growth factor-β1 (TGF-β1) that contributes to cancer progression. And CAFs are able to secret a broad spectrum of MMPs and, in turn, induce the remodeling of TME.38 Here, we investigated the effect of the preparations on NIH/3T3 fibroblasts co-cultured with 4T1 cells in a transwell device in terms of α- smooth muscle actin (α-SMA, a marker of CAFs), tenascin C (TNC, a kind of protein secreted by CAFs) as well as TGF-β1 that would induce the activation. CLSM examination showed that the expression of α-SMA from the treatment with HNPs+HT was significantly less than that from other treatments (Figure 4F). The treatment with MATT-LTSLs+HT also markedly inhibited the expression of α-SMA on NIH/3T3 fibroblasts. Western blot assay demonstrated that the expressions of TGF-β1 and TNC were significantly reduced by the treatment with HNPs followed by MATT-LTSLs (Figure 4G). Further quantitative analysis showed that the treatment of HNPs+HT enabled approximately 10-fold reduction in the expressions of TGF-β1 and TNC compared with that of the saline treatment (Figure 4H, I). Overall, these results demonstrated that 12


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the HNPs markedly suppressed the activation of fibroblasts. Invasion inhibition. Mortality in breast cancer patients mainly results from the metastasis of cancer cells.25 To investigate the ability of HNPs to inhibit the metastasis of 4T1 cells, a cell invasion experiment was performed using a Matrigel Transwell assay. In contrast to the control, the three formulations suppressed the migration of 4T1 cells. In particular, MATT-LTSLs caused significant inhibition compared to HA-PTX, although MATT did not lead to cytotoxicity. Importantly, the combined formulation, HNPs, further reduced the migration and demonstrated synergy (Supplementary Figure S9A, B). In

vivo

tumor

targeting

and

biodistribution.

(1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine

In

iodide),

contrast both

to

free

DiR

DiR-LTSLs

and

HA-PTX/DiR-LTSL HNPs achieved marked tumor accumulation with increasing accumulation over time in a period from 1 to 24 h (Figure 5A). Importantly, the fluorescence signal from HA-PTX/DiR-LTSL HNPs in isolated tumor tissues collected at 24 h postinjection was significantly stronger than that from DiR-LTSLs (P < 0.01) (Figure 5B, C), which demonstrated that the coating of HA on LTSLs improved the tumor targeting of the nanoplatform. To study the co-delivery of HA-PTX and DiR-LTSLs to the tumor site, dual-labeled Rho-HA-PTX/CF-LTSL HNPs were injected into mice, and tumor tissues were harvested 2 h after injection to prepare frozen sections for CLSM examination. As depicted in Figure 5D and E, yellow fluorescence was easily identified after merging the red fluorescence (Rho-HA-PTX) and green fluorescence (CF-LTSLs) images. Therefore, this result indicated that the HNPs successfully co-delivered Rho-HA-PTX and CF-LTSLs into the tumor tissue and were stable in the circulation system without disintegrating for the absorption of plasma protein. 13


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Next, we investigated the location of these nanoparticles inside tumors; frozen tumor sections were prepared using a similar procedure 2 h after treatments with CF-LTSLs+HT, FITC-HA-PTX, FITC-HA-PTX/LTSL HNPs+HT, or HA-PTX/CF-LTSL HNPs+HT. The co-localization of these nanoparticles with microvessels inside tumors demonstrated that these nanoparticles penetrated the tumors (Figure 5F–I). Additionally, the prodrug HA-PTX accomplished excellent penetration and cell entry (Figure 5G). Importantly, unlike the results seen in Figure 5D without HT treatment, a large amount of green fluorescence in the pre-nucleus regions was displayed after local HT treatment. These results suggested that HA-PTX/CF-LTSL HNPs penetrated the tumor tissue well and that the release of their payloads, HA-PTX and CF, was accomplished by HT treatment. The payloads then crossed the microvessel and either subsequently entered the cancer cells or remained in the ECM. In vivo antitumor efficacy. First, the effect of MATT dose on antitumor activities was evaluated. As shown in Supplementary Figure S10, the treatment with MATT-LTSLs at MATT doses of 5 mg/kg and 10 mg/kg inhibited the tumor-growth with extremely higher efficiency than the treatment at 2.5 mg/kg. Hence, these two doses were employed for further treatment. Next, we studied the influence of different preparations on antitumor effects. The tumor volume from the groups treated with control formulations for 18 days increased by 20–30-fold compared to that on the first day of treatment (Figure 6A). In contrast, the tumor volume in the groups treated with HNPs+HT at different doses was approximately 6-fold higher than the initial volume. The body weights of the mice were either unaltered or the mice regained their body weight after treatment with HNPs+HT at different doses, indicating that these formulations were safe (Figure 6B). Additionally, a 12-fold change in tumor volume observed in the free PTX+MATT group at the end of treatment was 14


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significantly smaller than that observed in the free PTX or MATT group, both of which exhibited an approximately 18-fold increase in tumor volume. These results were confirmed by examining the size and weight of isolated tumor tissues (Figure 6C, D). The antitumor activities of these formulations were further studied in tumor tissues after administration was completed. As shown in histological sections (Figure 6E), the saline and HT groups had few necrotic cancer cells. In contrast, the formulations for free PTX and MATT, HA-PTX, MATT-LTSLs and PTX+MATT displayed moderate necrosis. HNPs+HT groups showed serious necrosis with distinct alternations in morphology compared to the controls. A TUNEL assay revealed that the percentage of apoptotic cells (brown) in the HNPs+HT groups, which was as high as 75%, was significantly greater than that in the single (free) drug groups and the other control groups (Figure 6F, H). Ki67 analysis further confirmed the results of the TUNEL assay (Figure 6G, I). The combination of free PTX and MATT had enhanced antitumor efficacy compared with their isolated use, demonstrating their synergy, and crucially, the antitumor effect was significantly improved due to the co-delivery of HA-PTX and MATT-LTSLs using HNPs. In vivo metastasis and angiogenesis. MMPs are the principal enzymes for the decomposition of the extracellular matrix; therefore, they play an essential role in the invasive growth, metastasis and angiogenesis of cancer and in various chronic inflammatory diseases.39 Thus, investigations of angiogenesis and lung metastasis were performed at the end of the experiment. In the saline and HT groups, microvessels (brown) inside the tumor were found throughout (Figure 7A) with a microvascular density (MVD) greater than 170 vessels/field (Figure 7B). The number of microvessels in the groups treated with drug-containing formulations was dramatically reduced compared to that in the control groups. The MVD in HA-PTX or MATT-LTSL groups was significantly less than that in 15


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PTX and MATT groups and in the group treated with free PTX+MATT. In particular, the groups treated with HNPs, irrespective of the MATT dose, displayed few microvessels with a MVD less than 10. Figure 7C shows the lung metastasis of breast cancer in 4T1 tumor-bearing mice. More than 20 metastatic nodules with considerable size were displayed in the saline and HT groups (Figure 7D). In contrast, the treatments with drug-containing formulations caused significant declines in the number of nodules. Interestingly, MATT-LTSLs exhibited markedly improved metastasis inhibition compared with free MATT, thereby indicating that LTSLs are capable of delivering MATT to a tumor site with high efficiency. Most importantly, the groups treated with HNPs showed no nodules, and accordingly, HNPs had overwhelming advantages in metastasis inhibition compared to other formulations. These results demonstrated that only the co-delivery of HA-PTX and MATT by HNPs would have the most profound synergistic effects for angiogenesis and metastasis inhibition. In vivo inhibition of MMP expression and activity. MMPs, especially MMP-2 and MMP-9, are known to be involved in both angiogenesis and tumor metastasis. To explore the mechanism through which HNPs inhibited metastasis, gelatin zymography on collected tumor tissues from 4T1 tumor-bearing mice on post-injection day 19 was performed to examine the activity of both MMP-2 and MMP-9. The gelatinase activity of MMP-2 (72 kd) and MMP-9 (92 kd) is depicted in Figure 8A. In gelatin zymography, bright gel bands indicating MMP activity appear when gelatin is enzymatically decomposed by MMP-2 and MMP-9.40 Distinguishable bright bands, which suggest high activity for MMP-2 and MMP-9, were found in saline and HT groups. After treatment with drug-containing formulations, the MMP-2 and MMP-9 activity was reduced, as evidenced by the decreased area and brightness of the bands. Among these groups, the group treated with HNPs (10 mg/kg, 10 mg/kg) 16


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displayed the lowest gelatinase activity, and further quantification indicated that the MMP-2 and MMP-9 activity decreased by 4.5-fold and 4.3-fold, respectively, compared to the saline group (Figure 8B). These results demonstrated that MMP activity was dramatically inhibited by HNPs at a fixed dose. Next, MMP-2 and MMP-9 expression were detected by a western blot assay (Figure 8C). In saline and HT groups, high expression of MMP-2 and MMP-9 protein was displayed. In contrast, the treatments with these drug-containing formulations significantly decreased the expression of MMP-2 and MMP-9 (Figure 8D, E). Noticeably, HNP treatment, especially at high doses (10 mg/kg, 10 mg/kg), significantly reduced the expression of MMP-2 and MMP-9. The expression of MMP-2 and MMP-9 after HNP treatment at this dose was approximately 3 and 4, 4 and 5, 4 and 6, 5 and 7, and 5 and 8-fold lower than that after MATT+PTX, MATT-LTSL, HA-PTX, MATT, and PTX treatment, respectively, demonstrating that the co-delivery of MATT and HA-PTX markedly suppressed the expression of MMPs. In vivo inhibition of fibroblast activation. To examine the expression of α-SMA, TNC and TGF-β1 in tumors after treatment, immunochemistry staining and western blot assay for the tumors collected at the end of experiment were performed. The treatment with HNPs at the three doses greatly suppressed the expression of both α-SMA and TNC in tumors, followed by the treatment with MATT-LTSLs (Figure 8F, G). Western blot assay displayed that the expression of TGF-β1 in tumors treated with HNPs, especially at the MATT/PTX dose of 10/10 mg/kg, was significantly lower than that from the treatment with other formulations (Figure 8H). Quantified analysis demonstrated that the HNP treatment at this dose reduced the TGF-β1 expression by 5-fold compared with the saline treatment (Figure 8I). These data indicated that HNP treatment efficiently inhibited the activation of 17


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fibroblasts in tumor. In vivo inhibition of ECM degradation. We hypothesized that the inhibition of MMP by HNPs would reduce the decomposition of ECM by MMPs. Here, the ECM components including fibronectin (FN), laminin (LN) and collagen, which are the substrates of MMPs,41 were measured by immunochemistry staining or Masson Trichrome staining after treatment. The level of FN in the tumors treated with HNPs at three doses or MATT-LTSLs at a MATT dose of 10 mg/kg was higher than that of the treatment with other formulations (Supplementary Figure S11A). The treatment with the MATT-loading preparations upregulated the LN level, with a higher expression from the HNP administration at PTX/MATT doses of 10/10 mg/kg and 5/10 mg/kg (Supplementary Figure S11B). Furthermore, the collagen content from HNPs was the highest among the formulations (Supplementary Figure S11C). Overall, these results demonstrated that the administration of HNPs inhibited the degradation of the major ECM components. In vivo safety evaluation. To evaluate the safety of HNPs, healthy mice were injected with LTSLs, HA-ADH, HA-PTX, MATT-LTSLs and HNPs every three days 5 times. CD68, a lysosomal protein that is present in activated macrophages, was monitored to assess immune response.42 As shown in CD68 immunochemistry (Supplementary Figure S12A), there were no positive cells in sections of heart, liver, spleen, lung, or kidney treated with HA-ADH or LTSLs, as seen in the control group (saline). This result indicated that HA-ADH and LTSLs did not cause toxicity. Histological analysis indicated that the injection of HNPs also did not induce pathological damage in these organs (Supplementary Figure S12B). No apparent signs of dehydration, muscle loss, locomotor impairment, or other symptoms associated with systemic toxicity were observed. Therefore, HNPs can be considered safe for injection. 18


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DISCUSSION A multifunctional nanoplatform self-assembled from liposomes and an HA-based prodrug is developed. In general, to obtain CD44-receptor mediated targeting, HA can be used to coat cationic liposomes via electrostatic interaction, or it can be covalently coupled to the polar headgroup of the lipid.43, 44 Few reports have indicated that an HA-based prodrug with negative charge is capable of directly assembling on liposomes, especially negatively charged liposomes. As indicated in the study of FRET, the HA-PTX prodrug was able to assemble on positively charged liposomes, LTSLs and LTDSLs and on liposomes with negative surface potential such as SSLs. Moreover, the measurement of DPH anisotropy demonstrated that the assembly induced 15%–27% reduction in the membrane fluidity of liposomes, in turn confirming the coating of HA-PTX. A previous report indicated that the membrane fluidity of liposomes was decreased by approximately 8%–12% by incorporating a hydrophobic peptide into the lipid layers at lipid/peptide mass ratios of 600:1–200:1 during preparation.33 On the contrary, the insertion of a bile salt into the lipid bilayers at lipid/bile salt mass ratios of 7:1–3:1 allowed for 14%–47% increase in the membrane fluidity of liposomes.34 These reports implied that, to some extent, only when a material was incorporated in the lipid bilayers, would the membrane fluidity be altered. Additionally, via hydrophobic forces, certain amphiphilic substances or inorganic nanoparticles can be absorbed on lipid bilayers in aqueous conditions.45-47 A recent report revealed that increasing the hydrophobicity of a polymer increased the affinity for cell membrane interaction.48 And the hydrophobic force between lipid membranes and negatively charged biomaterials could overcome the electrostatic repulsion and thus render the material capable of insertion into the membranes.49 In this study, the grafting of PTX (a hydrophobic anticancer agent) endowed HA have hydrophobic chains 19


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and consequently increased the hydrophobicity of HA. Therefore, we speculate that the hydrophobic interactions enable the HA-based prodrugs and liposomes to hold together and, subsequently, the hydrophobic regions in the prodrug insert into the lipid bilayers, finally forming HNPs. This insertion was further verified by the stability study, which if little incorporation occurred the HNPs was unable to keep their stability in 10% FBS medium. Indeed, our further study demonstrated that another HA-based prodrug, HA-oridonin (ORD) that was prepared by conjugating the hydrophobic ORD with HA, could also assemble on liposomes, obtaining another type of HNPs. Here, we report that by assembling a prodrug consisting of a hydrophobic compound and polymers on liposomes, in particular on liposomes with negative charges, the development of a smart nanoplatform, which can co-load two or more active drugs for combined treatment via dual targeting, is feasible. In addition, the knowledge gained from coating an active targeted prodrug on the surface will help improve the medical performance of liposomes. HNPs can efficiently accumulate in tumor tissue and deeply penetrate tumors. Nanomedicines can accumulate in tumor tissue via an enhanced permeability and retention (EPR) effect. Nevertheless, poor penetration causes encapsulated drugs to be released only in the perivascular space of tumor areas without accessing hypoxic tumor cells that are not always sensitive to chemo- and radiotherapies.50, 51 In our study, unlike CF-LTSLs, the decoration with HA-PTX, which can specifically bind to CD44 receptors, significantly enhanced the tumor accumulation of HNPs in 4T1 tumor-bearing animal models (Figure 5C) and thereby endowed HNPs with enhanced tumor targeting. These results thus indicated that the surface modification of HA-based prodrugs could improve the tumor-targeting capacity of nanoparticles. Importantly, it was found that the HNPs with a diameter of 96-nm 20


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successfully penetrated the inside of the tumor (Figure 5F–I). Initially, previous investigations indicated that approximately 100-nm liposomes with or without surface decoration could penetrate the inside of the tumor.52, 53 A recent study also indicated that the size of the nanomedicine played a critical role in tumor penetration.54 Mild hyperthermia (41–42 °C) also modified the tumor environment by increasing tumor blood flow, oxygenation and vascular permeability, which improved drug delivery to the tumor.55 Two payloads encapsulated in HNPs can be released by HT treatment and be thus enabled to cross the tumor blood vessels and distribute throughout the tumor tissue. Tumor vascular normalization characterized in terms of a significant reduction in vascular permeability and vessel size hinders the nanoparticles’ ability to diffuse into the tumor tissue.51, 56 However, nanoparticles < 12 nm can diffuse into tumor tissue upon the normalization of tumor blood vessels.57 Therefore, it was desired that large nanoparticles > 100 nm would control the release of their payloads or change to ultrasmall nanoparticles < 12 nm within the blood vessels in tumors.58, 59 In this study, to achieve synergistic therapy, MATT was included to target extracellular MMPs located in the TME, while HA-PTX was chosen to achieve intracellular delivery. LTSLs, after local HT tumor treatment, can rapidly release their payloads within microvessels where they subsequently penetrate tumors.55 As a result, LTSLs were chosen as carriers for these two active agents. From in vitro experiments, it was evident that HNPs released their payloads quickly after HT treatment at 42 °C (Figure 2H), and the released HA-PTX rapidly entered the cancer cells via CD44 receptor mediation (Figure 3). Additionally, the in vivo results revealed that the released payloads diffused into the tumor tissue and distributed throughout the tumor in large quantities (Figure 5H, I). The intratumoral distribution of these two payloads could be taken up by cells or remain in the matrix network. Accordingly, these HNPs have the potential for 21


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dual targeting of the TME and tumor cells, and they can therefore enhance the therapeutic efficacy of drugs. MATT, a matrix metalloproteinase inhibitor, displayed modest efficacy in delaying disease progression in preclinical and clinical studies. However, due to the cumulative toxicity from inflammation and musculoskeletal pain, its Phase III study was cancelled.5, 60 These side effects might stem from the broad-spectrum inhibitory activity of MATT and the inhibition of disintegrin and metalloproteinase family members as well as aggrecanases.61 In contrast with free MATT, MATT-LTSLs accumulated significantly in tumors and showed marked improvement in the inhibition of tumor growth, angiogenesis and metastasis (Figures 6, 7). However, MATT-LTSLs were more potent in the suppression of angiogenesis and metastasis compared with the tumor-growth inhibition. Interestingly, MATT-LTSLs had significant toxicity against 4T1 cells, although free MATT was not cytotoxic (Figure 4B, E). In addition to degrading components of the ECM, MMP function interferes with the induction of apoptosis of cancer cells by cleaving ligands or receptors that transduce pro-apoptotic signals.3 In general, MMPs distribute in the ECM, but there are also certain MMPs such as membrane-type MMPs (MT-MMPs) present in the cell membrane.3, 62-64 The efficient delivery of MATT to the TME with the LTSLs increases the chance for MATT to bind to MT-MMPs and consequently promote the transduction of proapoptotic signals. This mechanism explains why MATT-LTSLs caused profound cell apoptosis and warrant further study. Regardless, LTSLs are a promising delivery system for improving the therapeutic efficacy of MATT; and MATT, particularly MATT-LTSLs enable robust inhibition of angiogenesis and metastasis. Keeping TME’s integrity is an efficient approach for metastasis inhibition. Previous reports by 22


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other groups indicated that destroying the TME could improve the penetration of nanoparticles into tumor tissues and enhanced their antitumor efficacy.65-68 Nonetheless, the destruction of the TME naturally drives the cancer cell to escape from their broken “home (TME)” and facilitates the metastasis. In this case, an approach, which can simultaneously protect the TME from being destructed to “lock” the cancer cells in their “home” and kill the cancer cells, might be a promising strategy for curing highly metastatic breast cancer. MMPs decompose the ECM in TME and are therefore essential for tumors to metastasize throughout the body. Thus, suppressing the activity and expression of MMPs may protect the ECM against degradation and, therefore, keep the TME’s integrity. Here HNP treatment stopped the fibroblasts, one of the predominant component of stromal cells that supports the TME, being activated to CAFs by reducing the expression of TGF-β1 (Figures 4, 8) and, also, decreased the degradation of the major components in ECM (FN, LN, gelatin and collagen) by suppressing the MMP activity (Supplementary Figure S11), implying the scaffolding of TME was maintained without destruction. Furthermore, the treatment with HNPs as well as MATT-LTSLs allowed for profound inhibition of angiogenesis and metastasis (Figure 7). As a result, maintaining the TME’s integrity is able to significantly inhibit the metastasis. To our knowledge, such an approach for metastatic inhibition has not been reported previously. We believe that this findings offer opportunities for understanding the functions of the TME and would assist with rational design of a drug delivery system to treat metastatic cancer. Combined therapy with HA-PTX and MATT in HNPs allows for the robust treatment of metastatic breast cancer. The failure of cancer treatments is generally due to drug resistance and the resulting metastasis. As a result, the direct killing of tumor cells with a chemotherapy drug is not the 23


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most effective way to treat cancer. Combination treatment for metastatic cancer has many benefits, including enhanced efficacy, improved metasatic inhibition, reduced drug resistance, and reduced side effects. In this study, an efficient route for combined treatment for breast cancer was developed in which a nanosystem was designed to target the TME to maintain rather than break its integrity by inhibiting MMPs with MATT and thus reduce the metastasis of cancer cells and enhance the delivery of an HA-PTX prodrug via CD44 mediation to kill the cancer cells. The improved efficacy after HNPs dosing was demonstrated through the following results: (1) improved cytotoxicity against cancer cells; (2) efficient antitumor effects; (3) potent metastasis and angiogenesis inhibition; (4) significant suppression of MMP expression and activities; (5) profound inhibition of the fibroblast activation. The improved antitumor effect was mainly ascribed to the following mechanisms. Initially, there was a synergistic effect between HA-PTX and MATT that might promote cytotoxicity against cancer cells. Second, HNPs possessed the capacity for deep tumor penetration, and their released payloads triggered by HT treatment distributed in the entire tumor. Third, MATT-LTSLs enhanced the inhibition of MMP expression and activity, which greatly contributed to the maintenance of TME’s integrity and the resulting antimetastatic effects. Fourth, HNPs blocked the fibroblast activation by downreulating the TGF-β1 expression in tumor. Additionally, HA-PTX targeted CD44 receptors and therefore enhanced the cellular uptake of PTX. Noticeably, due to the excellent drug-loading ability of liposomes and the easy modification of HA by other active compounds, HNPs could also be a promising delivery system for the treatment of other diseases. Further work regarding co-delivery of protein drugs and HA-based prodrugs with the HNPs for immunotherapy is currently underway.

CONCLUSIONS 24


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In summary, this study offered a proof of concept that prodrugs of an HA-hydrophobic active compound and liposomes can be assembled into a multifunctional nanoplatform for the dual targeting of the TME and cancer cells and that the combined use of HA-PTX and MATT with the HNPs (~100 nm) is a promising approach for the treatment of metastatic breast cancer. The HNPs have several advantages, including the dual targeting of the TME and cancer cells, deep tumor penetration, the quick release of payloads, efficient diffusion into tumors and the ability to combine treatments for other diseases. This work provides avenues for preparing multifunctional nanoparticles and provides a cancer treatment strategy that maintains rather than breaks the TME’s integrity while combining other chemotherapy treatments. To our knowledge, the approach that inhibits the metastasis via keeping the TME’s integrity has not been reported previously. This finding brings a chance for understanding the complicated functions of the TME and would benefit the rational design of nanomedicine to treat metastatic cancer. Additionally, by tailoring the synergistic effects of its payloads, this nanoplatform has great potential applications for treating other diseases. Further work on co-delivery of protein drugs and HA-based prodrugs with the platform for immunotherapy is ongoing. In conclusion, a prodrug of HA-hydrophobic compound and liposomes are able to be assembled into a multifunctional nanoplatform that can simultaneously target the TME and cancer cells and greatly facilitate the combined therapy. We believe that other prodrugs composed of a water-soluble polymer and an insoluble drug could also be assembled on liposomes to prepare potent drug delivery system. Further work is currently in progress.

EXPERIMENTAL SECTION Materials. PTX with more than 98% purity was purchased from Yew Biotechnology Co. Ltd. 25


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(Jiangsu, China). Taxol (marked product of PTX) was purchased from Bristol-Myers Squibb Investment Co. Ltd. (Shanghai, China). MATT was purchased from Nanjing Adooq Co., Ltd. (Jiangsu, China). FITC, CF, Rho, rhodamine B isothiocyanate (RITC) and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenyltetrazoliumromide (MTT) were obtained from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). DiR was purchased from Biotium, Inc. (Hayward, CA, USA). 1-stearoyl-2 hydroxy-sn-glycero-3-phosphocholine

(1-StePc),

1,2-dioleoyl-3-trimethylammonium-propane

(DOTAP), cholesterol were purchased from Shanghai AVT Pharmaceutical Technology Co., Ltd. (Shanghai,

China).

1,2-dipalmitoyl-DL-alpha-phosphatidylcholine

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene

(DPPC), glycol)-2000]

(DSPE-PEG2000), soybean phosphatidylcholine (S100PC) was purchased from Lipoid (Germany). 4T1, MCF-7, A549, and NIH/3T3 cells were purchased from Nanjing KeyGEN Biotech Co., Ltd. (Nanjing, China). Fetal bovine serum (FBS), RPMI-1640, DMEM, Trypsin and Penicillin-Streptomycin Solution were obtained from Wisent Inc. (Nanjing, China). DAPI and Annexin V-FITC/PI staining kit were obtained from the Beyotime Institute of Biotechnology (Haimen, China). Lyso-tracker green was purchased from Shanghai Yeasen Biotech Co., Ltd. (Shanghai, China). Cell cultures. The 4T1 and A549 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin streptomycin combination and kept at 37 °C under 5% CO2. MCF-7 and NIH/3T3 cells were cultured in DMEM medium with 10% fetal bovine serum and 1% penicillin streptomycin. The cells were harvested with trypsin and the cell suspension was used for experiments. Synthesis of prodrug HA-PTX and characterization. HA-PTX was synthesized according to the method reported by Luo and Prestwich.30 Synthesis of the prodrug HA-PTX was accomplished through 26


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HA-ADH and PTX-NHS (N-hydroxysuccinimide) coupling. In brief, HA-ADH was produced through a reaction between ADH and HA. After mixing HA (3 mg/mL) and ADH at a molar ratio of 1:40 and adjusting the pH to 4.75, a 4-fold volume of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (compared to HA) was added to the mixture and incubated at pH 4.75 for 24 h at 25 °C. The reaction was terminated by adjusting the pH to 7.0. The reacted sample was dialyzed in sequence against 0.1 M sodium chloride, 25% ethanol, and pure water, and then the sample was freeze dried. The molecular weight cut off (MWCO) of dialysis bag was 3,500 Da. PTX-NHS was formed by modifying PTX-semi with a 2`-ester containing an N-hydroxysuccinimide moiety. PTX-semi was prepared as follows. PTX was mixed with succinic anhydride (1.2 equiv.) in dichloromethane, and then the mixture was stirred for 72 h with the addition of a 10-fold excess of anhydrous pyridine and purified by column chromatography with acetic ether and n-hexane as eluents. After dissolving the prepared PTX-semi and N-hydroxysuccinimide diphenyl phosphate (SDPP) (1.5 equiv.) in acetonitrile with triethylamine (4 equiv.) and stirring for 24 h, the reaction product was eluted with acetic ether and n-hexane. PTX-NHS (2 equiv.) was mixed with HA-ADH and reacted for 24 h under stirring. Prior to the reaction, HA-ADH was dissolved in phosphate buffer solution (PBS) (pH 6.5) to form a concentration of 1 mg/mL, and PTX-NHS (2 equiv.) was dissolved in a N,N-dimethylformamide (DMF)/H2O (2:1, v/v) solution. After dialysis (MWCO 3,500 Da) in 50% ethanol and then pure water, the synthesized HA-PTX was freeze dried, and a white solid powder was obtained. FITC/Rho-HA-PTX was synthesized as follows. After placing dimethyl sulphoxide (DMSO) containing FITC or RITC in an HA-PTX solution at pH 9 and stirring for 24 h, the mixture was dialyzed to remove the unconjugated fluorescence probes and then freeze dried. 27


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HA-PTX was characterized by 1H NMR, IR and UV/Vis. 1H NMR (500 MHz, D2O), δ 7.27 (s, 1H, PTX), 4.27 (D2O), 3.03, 2.41 (s, 6H, repeating chain for HA), 2.13 (s, 8H, N-acetyl protons in HA); IR: 1153.2 cm-1, 1090.3 cm-1, 1061.0 cm-1 and 945.9 cm-1 (band of HA); 706.6 cm-1 (band of PTX); UV/Vis: λmax 227 nm (Supplementary Figure S1). Preparation of LTSLs and other liposomes. LTSLs composed of DPPC: 1-StePc: DSPE-PEG2000 = 86:10:4 (mass ratio) were prepared by a film hydration method. To obtain a thin lipid film, these phospholipids were dissolved in a solution of chloroform:methanol = 3:1 (v/v) and dried at 45 °C under vacuum. The film was then hydrated with pH 6.5 PBS containing MATT at 45 °C for 40 min. The film was treated by miniature ultrasonic probe and extruded through a membrane filter with a pore size of 0.22 μm, and the prepared MATT-LTSLs were ultrafiltered to remove free MATT. Rho-loaded LTDSLs (Rho-LTDSLs), Rho or DiR-loaded LTSLs (Rho, DiR-LTSLs), Rho-loaded SSLs (Rho-SSLs) and CF-loaded LTSLs (CF-LTSLs) were prepared by the same procedure. The LTDSLs had a lipid composition of DPPC: DOTAP: 1-StePc: DSPE-PEG2000 = 76:10:10:4 (mass ratio). The molar ratio of S100PC/cholesterol/DSPE-PEG2000 was 90:10:5 (molar ratio) for the preparation of SSLs. Preparation of HNPs. HNPs were prepared by dispersing HA-PTX in 5 mL of MATT-LTSLs at a HA/lipid mass ratio of 1: 4 and then vortexing for 30 s at r.t. FITC-HA-PTX/Rho-LTSL HNPs, FITC-HA-PTX/Rho-LTDSL HNPs, HA-PTX/DiR-LTSL HNPs, and FITC-HA-PTX/Rho-SSL HNPs were prepared with a similar procedure, except that HA-PTX was labeled with FITC and LTSLs, LTDSLs or SSLs were loaded with Rho or DiR in advance. Characterization. Size, PDI and the zeta-potential of samples were measured using a Malvern 28


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zetasizer (Brookhaven Instruments, Holtsville, NY) at 25 °C based on the principles of DLS. The morphology of samples was observed with a JEM-1230 TEM (Tokyo, Japan) at an acceleration voltage of 200 kV. Prior to TEM examination, these samples were stained with 2% (v/v) phosphotungstic acid for 5 min, a drop of the stained sample was deposited on the carbon mesh, and then the excess sample was removed with filter paper and dried at 25 °C. The drug content was detected with an LC-10AT HPLC system (Shimadzu, Japan). The HPLC conditions for the PTX determination were described in a previous report.69 The separation of MATT was performed at 30 °C using an ODS C18 column (4.6 mm × 250 mm, Diamonsil, China). The samples were eluted with a mobile phase of methanol/H2O (65/35, v/v, pH = 3) at a flow rate of 1 mL/min and monitored at 210 nm. FRET. FRET was conducted with two probes, FITC and Rho, as donor and acceptor, respectively. The probes were used to study the coating of HA-PTX on the liposomes. FITC-HA-PTX at a fixed concentration of 1 mg/mL was assembled with Rho-liposomes at different concentrations to form three assembled HNPs with FITC/Rho ratios of 2:1, 1:1 and 1:2. The three assembled HNPs included FITC-HA-PTX/Rho-LTSL HNPs, FITC-HA-PTX/Rho-LTDSL HNPs and FITC-HA-PTX/Rho-SSL HNPs. The emission spectra of these samples were recorded at r.t. with a fluorescence spectrometer (SHIMADZU RF-5301PC, Japan) with 450 nm (donor, FITC) as the excitation wavelength. The split width for the excitation and emission were 5 nm and 15 nm, respectively. To confirm the FRET results, CLSM observations were performed. Briefly, 4T1 cells (1×105) were seeded on a round glass cover slip for 48 h before treatment with FITC-HA-PTX/Rho-LTSL HNPs at different ratios for 1 h. The cells were washed with cold PBS 3 times and then observed by 29


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CLSM (LSM700, Carl Zeiss, Germany). Membrane fluidity determination. The membrane fluidity of LTSLs with and without HA-PTX coating was studied by fluorescence anisotropy determination as reported previously.33-34 In brief, LTSLs and HA-PTX/LTSL HNPs were prepared and then diluted to 0.05 mM lipid. DPH (1 mM in tetrahydrofuran) was mixed with LTSLs or HA-PTX/LTSL HNPs at a lipid/DPH ratio of 500:1 (v/v), shaken for 90 min at r.t. and left overnight. The fluorescence anisotropy of DPH in the samples was measured using a LS-55 luminescence spectrometer (Perkin Elmer, USA) at excitation and emission wavelengths of 361 and 425 nm, respectively. The measurement was conducted at 25 °C, 37 °C and 42°C, respectively. Fluorescence anisotropy was calculated following the equation automatically by the spectrometer: 𝑙𝑣 −𝐺𝑙𝑣

𝑟 = 𝑙𝑣 𝑉+2𝐺𝑙𝑣𝐻 𝑉

𝐻

(1)

Where r is fluorescence anisotropy, G is the correct factor specified by instrument, 𝑙𝑣𝑉 and 𝑙𝑣𝐻 are the emission intensity excited by vertically polarized light measured with parallel or perpendicular emission polarizer. Serum stability. One milliliter of FITC-HA-PTX/Rho-LTSL HNPs was mixed with 4 mL of 1640 medium containing 10% FBS and incubated in a shaker (SHA-C, Jintan, China) at a shaking speed of 75 rpm at 37 °C. At specific time intervals, the samples were withdrawn and added in a 96-well plate. FRET measurement was conducted using a microplate reader (POLARstar Omega, Germany). Temperature-triggered release. CF was used to detect the thermosensitivity of LTSLs. The drug release from each formulation was carried out by a dialysis method using a ZRS-8G release tester (Tianjin, China). In brief, 1.5 mL of CF-LTSLs or HA-PTX/CF-LTSL HNPs were added into a dialysis 30


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bag (MWCO 8,000–14,000 Da) and incubated in 100 mL of PBS solution (pH 6.5) at 37 °C or 42 °C for different times. The release of CF was determined by measuring the fluorescence intensity with a fluorescence spectrometer (ex: 492 nm, em: 515 nm). Expression of CD44 on 4T1 cells. CD44 expression on 4T1 cells was examined by flow cytometry (MACSQuant Analyzer 10, Germany) and CLSM. For flow cytometry, cells (1×106) were washed with blocking buffer, incubated with 2 μg of FITC-labeled anti-mouse CD44 antibody (Abcam, Britain) at 4 °C for 1 h, rinsed with PBS for 3 times and resuspended in 1 mL PBS. For confocal imaging, 2×105 cells were cultured on a round glass cover slip and incubated with 0.4 μg of FITC-labeled anti-mouse CD44 for 1 h, washed with cold PBS and fixed with 4% paraformaldehyde for 10 min before staining with DAPI for 10 min. Real-time CLSM. 4T1 cells (1×105) seeded on a round glass cover for 48 h were treated with Rho-HA-PTX/CF-LTSL HNPs at a dye concentration of 0.5 μg/mL. The cellular uptake at 42 °C was monitored by real-time CLSM (LSM700, Carl Zeiss, Germany). CD44-mediated targeted delivery of HA-PTX. 4T1 cells (1×105) were seeded in a 12-well plate and pre-cultured with 10 mg/mL of HA or PBS in advance. Then, the cells were incubated with Rho-HA-PTX at different Rho concentrations for 4 h at 37 °C, washed with cold PBS and resuspended in 500 μL of PBS for flow cytometry analysis. Intracellular location of HA-PTX. The intracellular location of Rho-HA-PTX was studied in 4T1 cells with confocal imaging. Cells (2×105) seeded on 12-mm round glass cover slip in advance for cell attachment were incubated with Rho-HA-PTX or free Rho at a Rho concentration of 500 ng/mL in serum-free medium for 4 h at 37 °C. After washing the cells with cold PBS three times, the cells were 31


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treated with 1 mL of Lyso-tracker green for 2 h to stain the late endosomes and lysosomes. The CLSM examination was conducted after the cells were fixed with 4% paraformaldehyde and stained with DAPI for 10 min. Endocytosis pathway. 4T1 cells were seeded in 12-well plates at a density of 1×105 cells per well and cultured for 48 h. The cells were pre-incubated with specific endocytosis inhibitors for 30 min at 37 °C, including Cyto-D (10 μg/mL), nystatin (10 μM), methyl-β-cyclodextrin (M-β-CD), CPZ (10 μg/mL), nocodazole (20 μM), monensin (200 nM) and NaN3+DG (10 mM NaN3, 50 mM DG). The pretreated cells were cultured with Rho-HA-PTX at Rho concentration of 0.5 μg/mL at 37 °C for 2 h and then subjected to flow cytometry analysis. In vitro cytotoxicity. The cells were seeded in 96-well plates at a density of 5×103 cells/well and grown for 24 h. And the cells were incubated with drug-containing formulations and other blank carriers at various concentrations for 48 h at 37 °C. Subsequently, the cells were incubated with 20 μL of MTT (5 mg/mL) for 4 h and 150 μL of DMSO, respectively. The absorbance of each well was measured at 570 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific, America). In vitro cell apoptosis. Cell apoptosis was detected by fluorescence microscopy (Olympus IX53, Japan) and an Annexin V-FITC/PI-staining kit (Beyotime Biotechnology, China). In brief, 4T1 cells were seeded in a 6-well plate at a density of 2×105 per well for attachment. Then, the cells were treated with different drug-loaded formulations for 48 h at a PTX concentration of 10 μg/mL or a MATT concentration of 5 μg/mL. After removing the culture medium, the cells were washed with cold PBS. The treated cells observed by fluorescence microscopy were incubated with DAPI for 10 min to stain the nucleus. Prior to flow cytometry analysis, the cells were suspended in 500 μL of binding buffer and 32


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then stained with Annexin V-FITC/PI according to the protocol. In vitro inhibition of fibroblasts. NIH/3T3 and 4T1 cells were co-cultured in a transwell device and then incubated in DMEM with 10% FBS for 48 h, which the 4T1 and NIH/3T3 cells were in the top chamber and in the bottom, respectively. Then the 4T1 cells were incubated with 1 mL of serum-free medium containing drug-loaded formulations at 37 °C at a PTX concentration of 5 μg/mL or a MATT concentration of 2.5 μg/mL. The HT treatment was performed in a water bath at 43 °C. The cells were harvested with trypsin after a 24-h incubation, followed by immunofluorescence staining for examining the α-SMA and western blot assay for the expression of TGF-β1 and TNC. Immunofluorescence staining. Cover slides of NIH/3T3 cells were fixed, rinsed with PBS, blocked with goat serum blocking buffer for 20 min, incubated with an anti-SMA primary antibody at 4 °C overnight, rinsed with PBS 3 times, cultured with Alexa fluor 555 conjugated secondary antibody at 37 °C for 40 min, and stained with DAPI for 5 min, respectively. Western blot. To collect protein, the harvested 4T1 cells, NIH/3T3 cells or the tumors were incubated in RIPA buffer on ice for 10–20 min, homogenized and centrifuged at 9,000 g for 10 min at 25 °C. Total protein was measured using a BCA protein assay kit (Beyotime, China) and separated on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The proteins were then transferred to a nitrocellulose membrane, which was incubated in blocking solution containing 5% skim milk powder at 37 °C for 2 h and rinsed with phosphate buffer solution containing Tween 20 (PBST) 2–3 times. Subsequently, the membrane was incubated with primary antibodies at 4 °C for 2 h and a secondary antibodies at 4 °C overnight. Finally, immunoreactive proteins were visualized, and images were acquired with an Odyssey Infrared Imaging System (LICOR Biotechnology, Lincoln, NE) after staining 33


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with a chemiluminescence kit (KeyGEN Biotech., China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize protein expression. Integrated optical density (IOD) was calculated using the gel optical density analysis software Gel pro 4.0. Transwell invasion assays. In vitro Transwell invasion assays were performed in 24-well Transwell chambers (Corning Life Sciences, Inc., America) containing filters with 8-μm pores coated with diluted Matrigel® (BD Biosciences, America). Briefly, 4T1 cells were seeded at a density of 1×105 cells/well in the upper chamber and cultured for 48 h. The cells were then incubated with 2 mL of serum-free medium containing drug-loaded formulations at 37 °C for 24 h at a PTX concentration of 1 μg/mL or a MATT concentration of 0.5 μg/mL. After a 24-h incubation, cells in the upper chamber were removed using a cotton swab; cells that migrated to the lower surface of the ﬁlter were fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet for 10 min. The stained cells were visualized by optical microscopy (Olympus IX53, Japan). Then, crystal violet was dissolved in 33% acetic acid, and subsequently, the OD ratio was measured at 570 nm using a microplate reader. In vivo tumor targeting and biodistribution. The animals used in the experiments received care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals. All animal experiments were performed in accordance with the protocol approved by the China Pharmaceutical University Institutional Animal Care and Use Committee. Suspended 4T1 cells (0.2 mL) were injected into the upper back of BALB/c mice (female, 18–22 g) subcutaneously at a density of 1×106 per mouse. Treatment began when the tumor volume reached 500 mm3. Two hundred microliters of DiR labelled nanoparticles was injected into the mice via the tail vein at a fixed DiR dose of 0.5 mg/kg based on the animal’s body weight. At 1, 2, 4, 6, 8, 12, and 24 h after injection, 34


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the treated mice were anesthetized, and fluorescence images were acquired with in vivo imaging system (In-Vivo FX PRO, Carestream, Canada). Twenty-four hours after injection, the mice were sacrificed to harvest the main organs (heart, liver, spleen, lung, kidney, and tumor). Fluorescence images were acquired, and the fluorescence intensity of these organs was measured ex vivo using an in vivo imaging system. The intratumoral distribution of Rho-HA-PTX/CF-LTSL HNPs, CF-LTSLs, FITC-HA-PTX, HA-PTX/CF-LTSL HNPs, and FITC-HA-PTX/LTSL HNPs was examined. Two hours after injection and following HT treatment, the mice were killed to isolate tumor tissue, which was prepared as frozen sections. After staining microvessels with Cy7-labeled anti-mouse CD31 antibody (Abcam, Britain) for 1 h, CLSM was performed to observe the location of these nanoparticles. In vivo antitumor efficacy. Tumor-bearing mice were divided into 10 groups (n = 6) and treated with 0.2 mL of different preparations. Once the injections were completed, HT treatment was performed by placing the tumor area inside a water bath at 42 °C and maintaining the animal in a steady position for 45 min. The body weight and tumor volume were calculated every 3 days during the treatment period. At the end of treatment (at 19 days), the mice were sacrificed to collect tumors and lung tissues for further experiments. Cell apoptosis and proliferation were detected using TUNEL and Ki67 kits (Beyotime, China), respectively. The isolated tumor tissue was fixed in 4% paraformaldehyde and embedded in paraffin to prepare sections of 5-µm thickness. Then, an immunohistochemistry test was performed according to the standard instructions. The sections were observed and quantified under an optical microscope (B1-330, Motic, China) in five representative fields to provide a final report. 35


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Angiogenesis was evaluated by an index of MVD described in previous reports.16, 70 The tumor sections (5 µm) prepared for immunohistochemistry were stained with CD31 to quantify the vessel density inside tumors. In brief, the paraffin-embedded sections were prepared for antigen retrieval after being dewaxed, rehydrated and microwaved. Then, the sections were pre-incubated in 3% hydrogen peroxide in methanol at r.t. for 10 min and incubated with 0.25% trypsin for 10 min. Immunohistochemical staining was performed overnight using a CD31 monoclonal antibody as a primary antibody and incubated with secondary antibodies for 30 min. After adding fresh diaminobenzidine (DAB) solution and incubating the samples with hematoxylin for 10 min, the slides were rinsed with distilled water. Five representative areas were observed using an optical microscope to quantify the number of microvascular areas. Immunochemistry staining for testing α-SMA, TNC, FN and LN was performed as the similar procedure except for the primary antibodies were a α-SMA primary antibody (proteintech, USA), a TNC primary antibody (BOSTER, China), a fibronectin primary antibody (Abcam, Britain) and a laminin primary antibody (BOSTER, China), respectively. For histological analysis, the tumors were cut into 5-µm-thick sections and treated with hematoxylin and eosin (H&E), and the sections were observed under an optical microscope. For metastatic evaluation, the harvested lungs were fixed in Bouin’s solution for 72 h at 25 °C, and the colonies appearing on the surface of the lungs were quantified to assess lung metastases from breast cancer. Gelatin zymography. Gelatin zymography was conducted as previously described by Ikeda et al.71 Briefly, 10 μL of proteins extracted from different groups of tumors were analyzed on 8% SDS-PAGE containing 0.4% gelatin. Next, the gels were rinsed twice with 2.5% Triton X-100, 36


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incubated in 8% separation solution for 4 h at r.t., stained with Coomassie Blue for 2 h and destained in 25% methanol for 1 h and 10% acetic acid for 1 h. Areas with clear bands on the dark background indicated gelatin degradation. Relative MMP activity was calculated from the ratio between the density of the active band and the total densities of the bands for the active and pro-MMP. Quantitative analysis of gelatinase activity from zymographs was performed with Gel pro 4.0. In vivo safety studies. Thirty-five normal BALB/c mice were randomly divided into 7 groups (5 animals/each) and treated with different formulations. Eighteen days after administration, the mice were sacrificed to isolate the major tissues for H&E and CD68 immunohistochemical analysis. Statistical analysis. Data are presented as the means ± s.e.m. Differences between the groups were assessed by one-way ANOVA, and a P value less than 0.05 was considered significant.

AUTHOR INFORMATION *Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] ORCID L.F. Yin: 0000-0002-2825-3136 W. He: 0000-0003-3075-3831 Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT 37


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This study was supported by the National Natural Science Foundation of China (No. 81402869 and 81473152), the Natural Science Foundation of Jiangsu Province (No. BK20140671), the Jiangsu Qing Lan Project (2014, 2016), and the Fundamental Research Funds for the Central Universities. We also thank M. Sun and X. Ma for technical support from the Cellular and Molecular Biology Center of China Pharmaceutical University.

ASSOCIATED CONTENT Supporting information available: Figures S1–S12 (pdf): IR, 1H NMR and UV determination of HA-PTX, fluorescence emission spectra of FRET on other related nanoparticles, CD44 expression of 4T1 cells, free fluorescence probe uptake by 4T1 cells, time- and energy-dependent cellular uptake of Rho-HA-PTX by 4T1 cells, internalization mechanism, determination of combination index, cytotoxicity of drug-free carriers in tumor cells, inhibition of cell invasion in vitro, antitumor activity of MATT-LTSLs at different doses, inhibition of ECM degradation and safety examination. Supplementary movie 1 (avi): Real-time CLSM of Rho-HA-PTX/CF-LTSL HNPs entering 4T1 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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42. Peters, C. M.; Jimenez-Andrade, J. M.; Jonas, B. M.; Sevcik, M. A.; Koewler, N. J.; Ghilardi, J. R.; Wong, G. Y.; Mantyh, P. W. Intravenous Paclitaxel Administration in the Rat Induces a Peripheral Sensory Neuropathy Characterized by Macrophage Infiltration and Injury to Sensory Neurons and Their Supporting Cells. Exp. Neurol. 2007, 203, 42-54. 43. Qhattal, H. S. S.; Liu, X. Characterization of CD44-Mediated Cancer Cell Uptake and Intracellular Distribution of Hyaluronan-Grafted Liposomes. Mol. Pharmaceutics. 2011, 8, 1233-1246. 44. Fan, Y.; Sahdev, P.; Ochyl, L. J.; J. Akerberg, J.; Moon, J. J. Cationic Liposome–Hyaluronic Acid Hybrid Nanoparticles for Intranasal Vaccination with Subunit Antigens. J. Controlled. Release 2015, 208, 121-129. 45. Palominos, M. A.; Vilches, D.; Bossel, E.; Soto-Arriaza, M. A. Interaction between Amphipathic Triblock Copolymers and L-Α-Dipalmitoyl Phosphatidylcholine Large Unilamellar Vesicles. Colloids Surf. B 2016, 148, 30-40. 46. Kostarelos, K.; Tadros, T. F.; Luckham, P. F. Physical Conjugation of (Tri-) Block Copolymers to Liposomes toward the Construction of Sterically Stabilized Vesicle Systems. Langmuir 1999, 15, 369-376. 47. Liu, J. Interfacing Zwitterionic Liposomes with Inorganic Nanomaterials: Surface Forces, Membrane Integrity, and Applications. Langmuir 2016, 32, 4393-4404. 48. Rajan, R.; Hayashi, F.; Nagashima, T.; Matsumura, K. Toward a Molecular Understanding of the Mechanism of Cryopreservation by Polyampholytes: Cell Membrane Interactions and Hydrophobicity. Biomacromolecules 2016, 17, 1882-1893. 49. Wu, L.; Zeng, L.; Jiang, X. Revealing the Nature of Interaction between Graphene Oxide and Lipid 44


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65. Zhang, L.; Wang, Y.; Yang, Y.; Liu, Y.; Ruan, S.; Zhang, Q.; Tai, X.; Chen, J.; Xia, T.; Qiu, Y. et al. High Tumor Penetration of Paclitaxel Loaded pH Sensitive Cleavable Liposomes by Depletion of Tumor Collagen I in Breast Cancer. ACS Appl. Mater. Interfaces 2015, 7, 9691-9701. 66. Miao, L.; Li, J.; Liu, Q.; Feng, R.; Das, M.; Lin, C. M.; Goodwin, T. J.; Dorosheva, O.; Liu, R.; Huang, L. Transient and Local Expression of Chemokine and Immune Checkpoint Traps To Treat Pancreatic Cancer. ACS Nano 2017, 11, 8690-8706. 67. Guo, S.; Lin, C. M.; Xu, Z.; Miao, L.; Wang, Y.; Huang, L. Co-delivery of Cisplatin and Rapamycin for Enhanced Anticancer Therapy through Synergistic Effects and Microenvironment Modulation. ACS Nano 2014, 8, 4996-5009. 68. Ji, T.; Lang, J.; Wang, J.; Cai, R.; Zhang, Y.; Qi, F.; Zhang, L.; Zhao, X.; Wu, W.; Hao, J. et al. Designing Liposomes To Suppress Extracellular Matrix Expression To Enhance Drug Penetration and Pancreatic Tumor Therapy. ACS Nano 2017, 11, 8668-8678. 69. Jin, Z.; Lv, Y.; Cao, H.; Yao, J.; Zhou, J.; He, W.; Yin, L. Core-Shell Nanocarriers with High Paclitaxel Loading for Passive and Active Targeting. Sci. Rep. 2016, 6, 27559. 70. Li, Y.; Wu, Y.; Huang, L.; Miao, L.; Zhou, J.; Satterlee, A. B.; Yao, J. Sigma Receptor-Mediated Targeted Delivery of Anti-Angiogenic Multifunctional Nanodrugs for Combination Tumor Therapy. J. Controlled. Release 2016, 228, 107-119. 71. Ikeda, M.; Maekawa R.; Tanaka, H.; Matsumoto, M.; Takeda, Y.; Tamura, Y.; Nemori, R.; Yoshioka, T. Inhibition of Gelatinolytic Activity in Tumor Tissues by Synthetic Matrix Metalloproteinase Inhibitor: Application of Film in situ Zymography. Clin. Cancer Res. 2000, 6, 3290-3296.

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Table and caption Table 1. Physical and chemical properties of key nanoparticles Particle size Nanoparticles

Surface charge PDI ±S.E.M

(nm) ±S.E.M

(mV) ±S.E.M

–

–

–5.73±2.92

MATT-LTSLs

90.20±0.70

0.23±0.01

3.09±2.30

HA-PTX/MATT-LTSL HNPs

95.60±1.10

0.24±0.01

–1.62±1.25

LTDSLs

102.10±1.20

0.24±0.01

10.41±2.02

HA-PTX/LTDSL HNPs

109.60±1.90

0.28±0.01

7.13±1.86

SSLs

82.40±0.90

0.19±0.01

–2.21±1.35

HA-PTX/SSL HNPs

90.30±1.40

0.23±0.01

–4.37±1.72

HA-PTX

The HA/lipid and PTX/MATT mass ratios in these HNPs was 1:4 and 1:1, respectively.

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Figures and captions

Figure 1. Schematic illustration of HNP preparation and intended mechanism for metastatic breast cancer treatment. The components of HNPs are MATT-LTSLs and the prodrug HA-PTX. HA-PTX was synthetized and self-assembled onto the surface of MATT-LTSLs to form spherical hybrid drug-loaded nanoparticles. When HNPs pass through the tumor tissue, the release of HA-PTX and MATT encapsulated in LTSLs can be triggered by local heating of the tumor site. The released MATT will bind with the MMPs in the TME to prevent the TME against destruction and block the metastasis of tumor cells, whereas the HA-PTX prodrug, via targeting the CD44 receptor of tumor cells, will undergo CD44-mediated endocytosis and thus kill the cancer cells.

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Figure 2. Characterization. (A) Size distributions of LTSLs and HNPs. (B) Fluorescence emission spectra of FITC-HA-PTX/Rho-LTSL HNPs with FITC/Rho mass ratios of 1:0, 2:1, 1:1, 1:2 and 0:1, respectively.

Excitation

wavelength:

450

nm.

(C)

Fluorescence

emission

spectra

of

FITC-HA-PTX/Rho-LTSLs with (red) and without (black) HT treatment at 42 °C. (D) TEM images. (E) Images of 4T1 cells after treating with FITC-HA-PTX/Rho-LTSL HNPs at ratios of 2:1, 1:1 and 1:2 (green: FITC, red: Rho) at 37 °C. The images were acquired by CLSM. The scale bar is 5 μm. (F) 50


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Fluorescence anisotropy of DPH in HNPs with HA/lipid mass ratios of 1:8, 1:4 and 1:2, respectively (mean ± s.e.m., n = 3, *P < 0.05). The control was LTSLs. The determination was performed at 25 °C, 37 °C and 42°C, respectively. An increase in anisotropy value indicates reduction in membrane fluidity of liposomes. (G) Serum stability of FITC-HA-PTX/Rho-LTSL HNPs in 10% FBS medium was studied by FRET at 37 °C within a 48-h period (mean ± s.e.m., n = 3). (H) In vitro release profile at 42 °C or 37 °C. Data were presented as mean ±s.e.m. (n = 3,*P < 0.05, **P < 0.01).

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Figure 3. Cellular uptake of Rho-HA-PTX via CD44 mediation. (A) Cellular uptake of Rho-HA-PTX/CF-LTSL HNPs with a Rho/CF mass ratio of 1:1 in 4T1 cells with or without HT treatment. Extracellular release of HA-PTX was triggered by HT treatment at 42 °C (green: CF, red: Rho). Images were acquired by real-time CLSM. The scale bar is 10 μm. (B) Concentration (Rho)-dependent uptake in 4T1 cells was measured by flow cytometry. Cellular uptake in 4T1 cells with or without pre-incubation with 10 mg/mL HA: (C) Flow cytometry analysis, (D) CLSM 52


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observation, and (E) concentration (Rho)-related uptake (mean ± s.e.m., n = 3, *P < 0.05). Rho-HA-PTX was incubated with the cells for 4 h at 37 °C at a Rho concentration of 500 ng/mL. In CLSM examination, the nuclei were stained by DAPI (red: Rho, blue: DAPI). The scale bar is 5 μm. (F) CLSM images of 4T1 cells after 1-h, 2-h and 4-h incubations at 37 °C with Rho-HA-PTX (red: Rho, 500 ng/mL). The late endosomes and lysosomes were stained by lyso-tracker green. The scale bar is 5 μm.

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Figure 4. Cytotoxicity and apoptosis in tumor cells and inhibition of fibroblast activation. Cell viability was assessed by an MTT assay at 37 °C. HNPs with a PTX/MATT ratio of 1:1 were used in these experiments. Cytotoxicity of (A) PTX and HA-PTX, (B) MATT and MATT-LTSLs, 54


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MATT-LTSLs+HT, (C) HA-PTX/MATT-LTSL HNPs, and HA-PTX/MATT-LTSL HNPs+HT in 4T1 cells after a 48-h incubation at different concentrations (mean ± s.e.m., n = 5, *P < 0.05, **P < 0.01). (D) Fluorescence images were taken through fluorescence microscopy and the nuclei were stained with DAPI. The scale bar is 20 μm. Normal cells displayed homogeneous chromatin and showed spherical bright fluorescence without segmentation and fragmentation. In contrast, apoptotic cells exhibited nuclear condensation and chromatin pyknosis. (E) The apoptosis rate of 4T1 cells was determined by Annexin V-FITC/PI staining. The lower-left, lower-right, upper-right and upper left quadrants represented the viable, early apoptotic, late apoptotic and dead cells, respectively. The incubation was performed at a PTX concentration of 10 μg/mL or a MATT concentration of 5 μg/mL for 48 h at 37 °C. (F) CLSM images of NIH/3T3 cells co-cultured with 4T1 cells in a transwell device after a 24-h incubation with different formulations at a PTX concentration of 5 μg/mL or a MATT concentration of 2.5 μg/mL at 37 °C. The α-SMA was stained red with α-SMA antibody and the nuclei were stained blue with DAPI. The scale bar is 20 μm. (G) Western blot analysis of TGF-β1 and TNC expression. GADPH was used as a loading control. Dark bands indicate the protein expression. Quantitative analysis of the expression of (H) TGF-β1 and (I) TNC (mean ±s.e.m., n = 3, *P < 0.05, **P < 0.01).

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Figure 5. Tumor targeting in vivo. (A) In vivo fluorescence imaging of the 4T1 tumor-bearing BALB/c mice after intravenous injection with free DiR, DiR-labeled LTSLs (DiR-LTSLs) and HA-PTX/DiR-LTSL HNPs at different time points. The dose of DiR was 0.5 mg/kg based on animal’s weight. (B) Ex vivo fluorescence images of important tissues collected from the mice 24 h after administration and (C) fluorescence intensity of the DiR signal (mean ± s.e.m., n = 3, **P < 0.01). Co-localization of (D) Rho-HA-PTX and CF-LTSLs in tumor sections using (E) free Rho/CF as control. Nuclei were stained with DAPI (green: CF, red: Rho, blue: DAPI). Yellow fluorescence indicates intact HNPs. Tumor sections were obtained from isolated tumor tissues harvested from mice 2 h after injection with Rho-HA-PTX/CF-LTSL HNPs. The scale bar is 20 μm (enlarged view, 10 μm). The intratumoral distribution of (F) CF-LTSLs, (G) FITC-HA-PTX (green), (H) FITC-HA-PTX/LTSL 56


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HNPs, and (I) HA-PTX/CF-LTSL HNPs was observed by CLSM. The dose of the dyes was 0.5 mg/kg according to animal’s weight. The tumor tissues were isolated at 2 h after injection. Microvessels were stained red with Cy7-labeled CD31 antibody, and nuclei were stained blue with DAPI. The microvessels inside tumors exhibited red fluorescence, whereas fluorescent probe-labeled nanoparticles exhibit green fluorescence. Yellow fluorescence indicates the co-localization of nanoparticles with microvessels. The scale bar is 20 μm (enlarged view, 10 μm).

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Figure 6. Antitumor activity. Free PTX, free MATT, a combination of free PTX and MATT, MATT-LTSLs, and HA-PTX were administered to 4T1 tumor-bearing mice via tail vein injections every 3 days at a PTX dose of 10 mg/kg or a MATT dose of 5 mg/kg; HNPs were injected into mice at three doses (10/5 mg/kg, 10/10 mg/kg and 5/10 mg/kg for PTX/MATT). Saline was used as a negative control, and the injection volume was 0.2 mL. HT treatment was performed at 42 °C for 45 min 58


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immediately after injection. (A) Tumor volume and (B) body weight changes of 4T1 tumor-bearing mice. A comparison of the final tumor volume between groups was performed on day 18 (n = 6, *P < 0.05, **P < 0.01). (C) Tumor weight and (D) a digital picture of tumor tissues collected from 4T1 tumor-bearing mice on day 19 (mean ± s.e.m., n = 6, *P < 0.05, **P < 0.01). (E) H&E analysis, (F) TUNEL and (G) Ki67 immunochemistry of tumor tissues. In H&E analysis, the nuclei were stained blue, and the cytoplasm and extracellular matrix are stained red. The scale bar is 50 μm. In TUNEL and Ki67 analyses, the positive cells were stained brown. The scale bar is 20 μm. Quantitative analysis of (H) cell apoptosis and (I) proliferation (mean ± s.e.m., n = 5, *P < 0.05, **P < 0.01). The cell apoptosis and proliferation rates were quantified by five representative fields of cell nuclei counted under an optical microscope.

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Figure 7. Inhibition of angiogenesis and lung metastasis. Tumor and lung tissues for these examinations were harvested from 4T1 tumor-bearing mice on day 19 after administration. (A) Histological comparison of microvascular density (MVD) in tumor tissues. CD31-positive microvessels are stained brown. The scale bar is 20 μm. (B) Quantitative analysis of MVD was performed by quantifying five representative fields of CD31-immunostained sections under an optical microscope (means ± s.e.m., n = 5, *P < 0.05, **P < 0.01). (C) Digital pictures of the lung tissues. The arrows indicate the metastatic nodules in lung tissues. (D) Quantitative analysis of tumor nodules on lungs was performed by counting (means ±s.e.m., n = 6, **P < 0.01).

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Figure 8. Inhibition of MMP expression and activities and activation of fibroblasts. (A) The MMP activity of pro-MMP9 (130 kd, 225 kd), MMP-2 (72 kd) and MMP-9 (92 kd) in tumor tissues was analyzed by gelatin zymography. Bands of enzymatic activity were visualized by negative staining with standard Coomassie brilliant blue dye solution. Bright bands on the dark background indicate MMP activity. (B) Quantitative analysis of gelatinase activity from zymograms using a computer-supported image analysis program (means ± s.e.m., n = 3, *P < 0.05, **P < 0.01). (C) Western blot analysis of 61


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MMP-2 and MMP-9 expression in tumor tissues. GADPH was used as a loading control. Dark bands indicate MMP expression. Quantitative analysis of the expression of (D) MMP-2 and (E) MMP-9 (means ± s.e.m., n = 3, *P < 0.05, **P < 0.01). Immunochemistry staining of (F) α-SMA and (G) TNC in tumor tissues. The positive areas were stained brown. The scale bar is 20 μm. (H) Western blot analysis of TGF-β1 expression in tumor tissues. GADPH was used as a loading control. Dark bands indicate TGF-β1 expression. (I) Quantitative analysis of the TGF-β1 expression (mean ± s.e.m., n = 3, *P < 0.05, **P < 0.01). Tumor tissues for these experiments were collected from 4T1 tumor-bearing mice on day 19 after injection.

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