Quick-Responsive Polymer-Based Thermosensitive Liposomes for

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Controlled Release and Delivery Systems

Quick-Responsive Polymer-Based Thermo-Sensitive Liposomes for Controlled Doxorubicin Release and Chemotherapy Yulin Mo, Hongliang Du, Binlong Chen, Dechun Liu, Qingqing Yin, Yue Yan, Zenghui Wang, Fangjie Wan, Tong Qi, Yaoqi Wang, Qiang Zhang, and Yiguang Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00343 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 6, 2019

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ACS Biomaterials Science & Engineering

Quick-Responsive

Polymer-Based

Thermo-Sensitive

Liposomes for Controlled Doxorubicin Release and Chemotherapy

Yulin Mo, Hongliang Du, Binlong Chen, Dechun Liu, Qingqing Yin, Yue Yan, Zenghui Wang, Fangjie Wan, Tong Qi, Yaoqi Wang, Qiang Zhang, Yiguang Wang*.

Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China

State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China

KEYWORDS: NIPAM, HPMA, polymer, thermo-sensitive liposomes, doxorubicin,

photothermal

ACS Paragon Plus Environment

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ABSTRACT: Thermo-sensitive liposomes (TSLs) have been widely investigated for controlled drug release at specific pathophysiological sites. Although excellent thermosensitivity under hyperthermia (HT) was already realized for TSLs, their in vivo stability under physiological temperature still remains challenging. To overcome this limitation, optimized polymer-based thermo-sensitive liposomes (P-TSLs) with good thermosensitivity as well as satisfactory in vivo stability were developed in this study for tumorspecific controlled delivery of doxorubicin (DOX). In particular, polymers including p(NIPAM-r-HPMA) and p(HPMA-r-APMA) were successfully synthesized based on reversible addition-fragmentation chain transfer (RAFT) technique. Next, thermosensitive polymer p(NIPAM-r-HPMA) was first proposed to be inserted into the lipid bilayer of TTSL by post-insertion method. The resulting P-TTSL had phase transition temperature (Tm) around 42°C and displayed excellent thermo-sensitivity under HT: nearly 70% of DOX was released within 1 min when only 1% p(NIPAM-r-HPMA) was incorporated. Moreover, its stability was maintained at 37 °C. Compared with TTSL, significantly higher cellular uptake of DOX under HT was noticed in P-TTSL, indicating a burst release of DOX at 42 °C. In addition, both in vitro tumor spheroids experiments


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and in vivo tumor slices demonstrated an enhanced DOX deep penetration when treated by P-TTSL under HT. To achieve in vivo imaging and local HT under NIR, p (HPMA-r-APMA) was labeled by Cy7.5 and co-inserted into TTSL and the best drug efficacy was observed in CY-P-TTSL with HT along with prolonged blood circulation time. We have further investigated the biocompatibility of the developed CY-P-TTSL and reduced cardiotoxicity was observed even under HT in comparison with free DOX, demonstrating it is a reliable thermo-sensitive drug carrier for improving drug stability and therapeutic efficacy.

1. Introduction

Since the structure of liposomes was firstly reported in 1964, it has led the development of nanomedicine in the field of cancer treatment for half a century, achieving several milestones.1-2 The first targeted liposomes were developed in 1980; and the first nanomedicine, Doxil®, approved by FDA in 1995, was also a liposomal formulation.3-4 Liposomes demonstrated many advantages over the traditional drugs such as long blood circulation ability in vivo, preferential accumulation at the tumor site


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and so on. However, the drug release from such liposomes is slow in tumor sites, leading to poor bioavailability and therapeutic efficacy.5 Therefore, various stimuliresponsive liposomes were developed to overcome this challenge. While most of these systems are still under preclinical stages, thermo-sensitive liposomes (TSLs) have been progressed to an advanced stage of clinical developments due to their unique properties.6 With hyperthermia (HT) treatment, delivery of TSLs to tumors are significantly enhanced in vivo. More importantly, TSLs would quickly release drug under HT and result in high intravascular concentration in tumor vessels, driving drugs into the deep tissues down the concentration gradient.7 This is essential for competing the heterogeneity in different cancers where the enhanced permeability and retention (EPR) effect might not work for nanoparticles, like the high interstitial fluid pressure (IFP) in solid tumors would lead to poor penetration.8-9 Among various TSLs, lysolipid-based thermo-sensitive liposomes (LTSL) was the first TSL into clinical trial due to their impressing thermo-sensitivity.10 Nearly 80% of doxorubicin (DOX) could be released from the LTSL in 20 s under 42 °C.11 However, the LTSL was not stable in vivo with the blood DOX retention time only of ∼1 h as a


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result of the inclusion of lysolipid and lack of cholesterol.12 By contrast, traditional thermo-sensitive liposomes (TTSLs) were too stable to release drug, only 40% of encapsulated DOX could be released in 30 min under 42 °C, which is bad for intravascular drug release due to the fact that there are only ~50 s for liposomes to pass a 2 cm tumor via the vasculature.13-14 To find a balance between LTSL and TTSL, polymer-based thermo-sensitive liposomes (P-TSL) were exploited. By attaching thermo-sensitive polymers onto the liposomal membrane (even non thermo-sensitive lipids), the phospholipid bilayer would become destabilized when the temperature passes through the low critical solution temperature (LCST) of polymer and result in content release.15 However, previous studies mostly used free radical polymerization to synthesize thermo-sensitive polymers, which could cause large polydispersity index (PDI) of molecular weight and weaken the thermo-responsive sharpness.16 Also, the polymer/lipid mass ratio was relatively high (50% ~ 100%),17-20 which might lead to unpredicted in vivo toxicity due to the immunogenicity of polymers and drug leakage caused by weakened carrier stability.21 In


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addition, few studies reported the in vivo data of P-TSL, which is more important for potential use in clinic. In this study, we synthesized poly(NIPAM-r-HPMA) and poly(HPMA-r-APMA) with low PDI based on RAFT technique in which N-isopropylacrylamide (NIPAM) is the monomer that widely used in the synthesis of thermo-sensitive polymers and N-(2-hydroxypropyl) methacrylamide (HPMA) is a hydrophilic biomaterial that applied broadly in clinical trials with good biosafety.6,

22

The LCST of poly(NIPAM-r-HPMA) could be well tuned by

adjusting the composition of two monomers before RAFT co-polymerization23, and with only 1% polymer inserted onto TTSL, formed p(NIPAM-r-HPMA)-based thermosensitive liposomes (P-TTSL) could achieve rapid release of drug under 42 °C while maintaining its stability at 37 °C.


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Scheme 1. A Schematic illustration of (a) polymer-based liposomes prepared by postinsertion method and (b) NIR-induced DOX release in tumor vessels and interstitial fluids for controlled chemotherapy.


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As depicted in the scheme 1, firstly, DOX-loaded TTSL was prepared by ammonium sulfate gradient method.24 Next, p(NIPAM-r-HPMA) and p(HPMA-r-APMA) were postinserted onto the lipid bilayer using their C12 end group. p(HPMA-r-APMA) conjugated with NIR probe Cyanine 7.5 (Cy7.5) was used for in vivo imaging and providing heat for p(NIPAM-r-HPMA). Upon exposure to NIR laser, the Cy7.5 group could absorb light energy and transform it to thermal energy,25-26 heating the surrounding environment. When the temperature reaches above the LCST of p(NIPAM-r-HPMA), polymers would compress into shrinked state from expanded state, which might cause pore formation on the bilayer and increase membrane fluidity, resulting in rapid release of loaded DOX.27 After intravenous (i.v.) injection, the polymer-based thermo-sensitive liposomes would passively accumulate in the tumor tissues and penetrate into tumor interstitial fluids due to their good in vivo circulation ability. Then, with NIR laser, a local controlled HT treatment could induce quick release of DOX from liposomes in tumor vessels and interstitial fluids, generating very high DOX concentration gradient and pushing drug into deep tumor tissues. Finally, much more DOX could be internalized by tumor cells and obtain better drug efficacy than traditional nanomedicines.

Collectively, this study


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highlights

that

p(NIPAM-r-HPMA)

and

p(HPMA-r-APMA)

are

promising

novel

biomaterials to make polymer-based thermo-sensitive liposomes for imaging-guided, quick-responsive controlled cancer chemotherapy.

2. Results and Discussions

2.1 Syntheses and Characterization of p(NIPAM-r-HPMA) and p(HPMA-r-APMA). According to previous studies, p(NIPAM-r-HPMA) could be synthesized by RAFT polymerization using a dithiobenzoate-terminated chain transfer agent (CTA).23 But in this work, dodecylsulfanylthio-terminated CTA was creatively used to prepare p(NIPAM-

r-HPMA) with two main advantages for P-TTSL preparation: First, this would benefit polymers with a dodecyl end group which has strong hydrophobicity and mimics the C18 two-tail structure of host lipid, making polymers intended to be attached to membrane.2829

Second, the hydrophobic anchors used for polymer fixation were proved to be more

efficient to trigger drug release in a narrow temperature region when they were attached via the end group compared with those randomly distributed along the polymer backbones.30


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As described in Figure S1, 2, p(NIPAM-r-HPMA) and p(HPMA-r-APMA) with small PDI were synthesized via RAFT polymerization respectively and the characteristics of the polymers are summarized in Table S1. p(NIPAM-r-HPMA) with various compositions ranging from 95:5 to 80:20 were synthesized and the LCST of p(NIPAM-rHPMA) was elevated with the increase of HPMA ratio in the copolymer, which was ascribed to the stronger hydrophilicity of HPMA (Figure S3).31 Since the LCST was near 42 °C when NIPAM/HPMA was at a composition of 95:5, consistent with the phase transition temperature (Tm) of TTSL (40.9~ 42.4 °C),32-33 thus p(NIPAM95-HPMA5) was selected to prepare P-TTSL in the following experiments to facilitate the relatively poor drug release of TTSL at 42 °C. 2.2. Preparation and Characterization of P-TTSL. P-TSLs were generally prepared by dissolving lipids and polymers together in organic solvents, and then forming thin film through solvent evaporation.16, 34-36 By this method, P-TSLs could have polymer chains on both surfaces of the membrane and achieve a more intensive drug release above LCST in comparison with P-TSLs only having polymer chains on the outer surface. However, this could lead to much stronger content


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leakage at body temperature due to the destabilization of lipid membrane.37 Moreover, the loss of ammonium sulfate gradients might be caused by this method before drug loading, resulting in poor DOX encapsulation.

Figure 1. Characterization of TTSL and P-TTSL (polymer/lipid=1%, wt./wt.): (a) Particle size distributions at pH 7.4. (b) Zeta potentials at pH 7.4. (c) UV−Vis spectra of aqueous TTSL

and

P-TTSL.

Post-insertion

method:

Effects

of

incubation

time

(polymer/lipid=200%, wt./wt.) (d) and polymer/lipid mass ratios (e) on polymer inserting efficiency. (f) Effect of inserting polymer/lipid mass ratios on the stability of DOX-loaded liposomes. Data are presented as mean ± s.d. (n = 3).


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To ensure the stability of P-TTSL in vivo, post-insertion method was used in this work to attach polymers onto TTSL after DOX loading,18,

38

and the physicochemical

properties of the developed P-TTSL were first characterized, including particle size, zeta potential and UV spectra (Figure 1a-c). The particle size of TTSL was initially 89.19 nm and increased to 97.79 nm after post-insertion, indicating the inclusion of hydrophilic polymer chains. The zeta potential of P-TTSL was decreased to ~ -10 mV in comparison with the neutral potential of TTSL in PBS 7.4, which may be caused by the deprotonation of the carboxyl group at the end of the polymer, also demonstrating successful insertion of polymers onto lipid films. UV spectra exhibited no difference of DOX absorbance after post-insertion, indicating the same state of DOX in the liposomal lumen. In detail, the influence of incubation time and polymer/lipid mass ratio on the inserting efficiency was studied. As shown in Figure 1d, the inserting efficiency increased along with incubation time, and kept nearly constant after 1 h. Thus, 1 h was selected for polymer incubation in the following experiments. It was also noticed that with the increasing percentage of polymer/lipid from 25% to 200% (wt./wt.), inserting efficiency


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was reduced slightly (Figure 1e), suggesting saturation of inserted polymer on the bilayer. Due to the detection limit of fluorescent signals, the influence of lower mass ratio on inserting efficiency was not analyzed. But a higher efficiency may be expected from the reduction in total polymer amount, which made it less likely to get overloaded on the film. Finally, the influence of inserting polymer/lipid mass ratio on the stability of DOX-loaded liposomes was studied. When polymer/lipid was less than 5%, no significant leakage of DOX was observed after post-insertion (Figure 1f).


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Figure 2. Thermo-sensitive properties of P-TTSL: (a) TEM images of P-TTSL before and after hyperthermia at 42 °C for 2 min. Scale bar: 100 nm. (b) Differential scanning calorimetry (DSC) graphs of TTSL and P-TTSL. (c) Appearance of P-TTSL with different amount of p(NIPAM-r-HPMA) under 37 °C and 42 °C. (d) Transmittance of TTSL and PTTSL (polymer/ lipid = 200%, wt./wt.) suspensions as a function of temperature. (e) Temperature-dependent change of diameter and PDI value of P-TTSL (polymer/lipid = 1%, wt./wt.).

After the basic evaluation of post-insertion method, the thermo-sensitive properties of P-TTSL were studied. Displayed by TEM, the morphology of P-TTSL kept intact after it was heated at 42 °C for 2 min and cooled to room temperature (Figure 2a), implying that the shrinkage of polymers under HT did not break lipid film apart. Also, the incorporation of p(NIPAM-r-HPMA) into TTSL didn’t influence its phase transition temperature (Figure 2b) since polymer also has a LCST around 42 °C. As for the appearance, P-TTSL with different amount of p(NIPAM-r-HPMA) all appeared transparent under 37 °C (Figure 2c), revealing no self-aggregation under body


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temperature. However, they showed different appearance when temperature is above the LCST. This is because NIPAM polymer-coated nanoparticles tend to shrink and contract in response to increasing temperature when the concentration of free NIPAM polymer is low in solution. In opposite, when the free polymer concentration was high, NIPAM-coated nanoparticles tend to cross-link with each other, leading to the formation of aggregates39. For this reason, when lower amount of polymers was included (25%) was incorporated, the bilayer was supersaturated, inducing lots of free polymers, and consequently lead to huge aggregation of P-TTSL under HT, sharply decreasing the transmittance (Figure 2d). Moreover, both particle size and PDI of P-TTSL started to largely vary when the temperature was elevated near its LCST at 42 °C (Figure 2e), verifying their good thermo-sensitivity.


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Figure 3. Drug release profiles of P-TTSL in PBS 7.4 at 37 °C (a) and 42 °C (b) with different polymer/lipid mass ratios. (c) Temperature-dependent release profiles of TTSL and P-TTSL in PBS 7.4, incubated for 2 min. Data are presented as mean ± s.d. (n = 3).

To further optimize the polymer/lipid mass ratio, aimed at achieving the best balance between thermo-sensitivity and stability, in vitro release experiments were carried out. 1%, 5%, 25%, 100%, 200% (wt./wt.) p(NIPAM-r-HPMA)-based TTSL were prepared and their release behaviors were studied at 37 °C (Figure 3a) and 42 °C (Figure 3b) respectively. Stronger destabilization effect was noticed when higher amount of p(NIPAM-r-HPMA) was inserted onto the lipid bilayer: When polymer/lipid mass ratio reached 200%, over 90% encapsulated DOX was released from P-TTSL in 1 min at 42 °C. Still, it was not stable under 37 °C: over 40% DOX was discharged within 10 min.


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For comparison, TTSL incorporated with 1% polymer showed good stability under physiological temperature and nearly 80% of DOX was still remaining in liposome after incubation for 1 h. Additionally, this formulation held good thermo-sensitivity under HT: over 65% DOX was released in 1 min and 75% within 5 min, compared with less than 5% for TTSL in 1 min. These results demonstrated that with only incorporating a very small amount of p(NIPAM-r-HPMA) (1%, wt./wt.) into the lipid of TTSL, their thermo-sensitivity under HT could be significantly enhanced and kept their stability slightly influenced. Based on these data, 1% p(NIPAM-r-HPMA) was determined to be involved in P-TTSL for subsequent experiments. In addition, temperature-dependent DOX release behavior of P-TTSL was also studied and the results are presented in Figure 3c: P-TTSL exhibited intensive drug release at 42 °C. It’s interesting to note that for both P-TTSL and TTSL, the release percentage started to decrease when the temperature was higher than the Tm. This might be attributed to a more disordered state on the bilayer when temperature reaches over the Tm, which causes diminished lipid permeability.40 2.3 In Vitro Cytotoxicity Assay and Cellular Uptake of P-TTSL


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The cytotoxicity of liposomal DOX (P-TTSL, TTSL) on 4T1 tumor cells in serumcontaining media was compared with that of free DOX at 37 °C and 42 °C respectively. MTT assay was performed to evaluate the viability of cell populations in diverse treatment groups and the results are shown in Figure 4a.

Figure 4. In vitro cytotoxicity, cellular uptake and penetration evaluation. (a) Cytotoxicity of free DOX (blue), TTSL (dark grey) and P-TTSL (red) with or without heating at 42 °C


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for 5 min. Mean ± s.d. (n = 6), ***P < 0.001. (b) Fluorescence intensity analysis from flow cytometry in 4T1 cells after incubation with free DOX, TTSL and P-TTSL with or without HT for 5 min. Mean ± s.d. (n = 3). *P < 0.05, **P < 0.01. (c) In vitro cellular uptake of DOX by 4T1 cells incubated with TTSL and P-TTSL at 42 °C for different time periods. Scale bar, 25 μm. (d) The fluorescence distribution as indicated sections (white segments) in (c), which shows the extent of colocalization between DOX and the nucleus at 30 min. (e) Representative CLSM Z-stack fluorescence images of 4T1 tumor spheroids after incubation with TTSL and P-TTSL for 6 h at 37 °C after initial HT for 2 min. Scale bar, 100 μm. (f) Semi-quantitative analysis of fluorescence intensity in (e) as a function of Z-stack distance. Mean ± s.d. (n = 3), **P < 0.01.

At 37 °C, free DOX presented strong cytotoxicity at a concentration over 5 μg/mL while both P-TTSL and TTSL had negligible influence on cell viability, suggesting their good stability in serum. In contrast, significant reduction in viability for all groups was observed at 42 °C, and P-TTSL showed stronger cytotoxicity than TTSL at a high concentration of 50 μg/mL. Moreover, no conspicuous differences were noticed


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between the cytotoxicity of P-TTSL and free DOX during all concentration groups under HT, signifying quick temperature-response of P-TTSL to LCST and thorough release of encapsulated DOX from drug carrier. Next, cellular uptake efficiency in 4T1 cells with various treatments was evaluated by flow cytometry (Figure 4b), and P-TTSL group displayed nearly two-fold DOX fluorescence intensity (F.I.) at 42 °C compared with that in 37 °C, resulting from the quick release of free DOX under HT. However, no obvious difference was noticed for TTSL due to their relatively poor thermo-sensitivity. Interestingly, a significant increase in the uptake of free DOX was also observed, probably caused by improved cell membrane permeability under HT.41 Furthermore, time-dependent cellular uptake of PTTSL at 42 °C and 37 °C were explored by confocal laser scanning microscope (CLSM). As exhibited in Figure 4c, P-TTSL displayed much more intensive intracellular distribution of DOX and co-localization with nuclei at all experimental time points than TTSL at 42 °C: The intracellular fluorescent signal of DOX became detectable at 15 min in P-TTSL while none was visible in TTSL simultaneously. Meanwhile, after heating for 30 min, nearly 3-fold DOX signal in nuclei was observed in P-TTSL group compared


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with TTSL (Figure 4d and S4). For comparison, no co-localization between DOX and nuclei was observed for both treatments without HT (Figure S5), confirming the stability of P-TTSL under body temperature. 2.4 In Vitro Penetration of Tumor Spheroid Tumor spheroid, as a novel tumor model in vitro, is widely used in recent studies to simulate in vivo tumor microenvironment and evaluate drug penetration against tumor barrier.42-43 Here, 3D tumor spheroids composed of 4T1 cells were constructed to display DOX penetration. After the tumor spheroids were well-formed, P-TTSL and TTSL solution preheated at 42 °C were added and incubated for 1 h and 6 h separately before image capture by CLSM. As shown in Figure 4e and S6a, more DOX deep penetrated into the core of spheroids in P-TTSL group in comparison with TTSL at all time points: When incubated for 1 h, DOX in TTSL group was mainly located on the periphery of the spheroids at scanning depths over 30 μm, whereas P-TTSL reaching closer to the core and having stronger F.I. at the same distance (Figure S6b). After 6 h’s incubation, free DOX fully penetrated into the core, resulting in over 2-fold total F.I. in PTTSL group compared with TTSL group at scanning depths over 10 μm (Figure 4e, f).


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These results indicate that by attaching thermo-sensitive p(NIPAM-r-HPMA) onto the phospholipids of TTSL, a higher DOX release could be achieved under HT and lead to deeper DOX tumor infiltration in vitro, which could be a strong support for in vivo tissue penetration experiments. 2.5 In Vivo Pharmacokinetics Study To monitor the in vivo behavior of P-TTSL under NIR light, Cy7.5 conjugated p(HPMA-r-APMA) were involved in the post-insertion process in addition to p(NIPAM-rHPMA). The physiochemical properties of the prepared CY-P-TTSL are shown in Figure S7 and Table S2, indicating successful inclusion of Cy7.5 polymers and stable DOX encapsulation. Also, to explore whether the introduction of p(HPMA-r-APMA)-Cy7.5 into P-TTSL would have influence on the its release profile and optimize the amount of inserted polymers, in vitro release experiments were carried out under 37 °C and 42 °C respectively (Figure S8a, b). When the mass ratio of Cy7.5/total lipid was 0.1%, no significant difference was noticed for their release profile at 1 h under 37 °C in comparison to those without Cy7.5 polymer. And the insertion of p(HPMA-r-APMA)Cy7.5 did not undermine the thermo-sensitivity under HT either. Hence, p(HPMA-r-


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APMA)-Cy7.5 at an amount of Cy7.5/total lipid = 0.1% was determined for in vivo experiments and the composition of different liposomes was summarized in Table S3. In addition, as depicted in Figure S8c, when applied to 808 nm laser, CY-P-TTSL could transfer light energy to thermal energy which was enough to provide heat for the surrounding p(NIPAM-r-HPMA) to get shrinked. IR-tuned DOX release of CY-P-TTSL was also studied (Figure S8d). Interestingly, a higher release percentage was noticed at the same temperature compared with that in water bath, which might be attributed to the transient elevation of local temperature over LCST under NIR laser.44-46 Finally, the stability during storage at 4 °C and serum stability under 37 °C were also studied (Figure S9), showing the inclusion of p(NIPAM-r-HPMA) and p(HPMA-r-APMA)-Cy7.5 did not weaken the stability of liposomal carrier. Prolonged blood circulation would enhance tumor accumulation of nanoparticles,47 therefore it’s important to ensure carrier stability in vivo. Although poor blood retention has long been a problem for thermo-sensitive liposomes (e.g. LTSL, 0.92 ± 0.17 h plasma half-life),48 TTSL has relatively better performance among them,49 due to the inclusion of cholesterol to membranes which greatly improves the mechanical properties


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of lipid and decrease permeability.50-51 Here, to verify whether TTSL still maintain their in

vivo stability after the incorporation of p(NIPAM-r-HPMA) and p(HPMA-r-APMA)-Cy7.5, the blood circulation profiles of CY-P-TTSL, TTSL and free DOX were studied, and the results were shown in Figure 5a. It’s observed that free DOX had extremely rapid blood clearance: Only 8.18 ± 3.30% injected dose was remaining in plasma 2 min postinjection, and this amount was lower than 1% after 15 min. In contrast, CY-P-TTSL and TTSL exhibited significantly better circulation ability in the blood. Approximately 68.02 ± 16.41% and 71.40 ± 9.59% DOX remained in blood circulation at 15 min after i.v. injection respectively, and 40.70 ± 20.69% stayed at 1 h for CY-P-TTSL, even slightly higher than that of TTSL (24.40 ± 9.68%). This difference might be caused by additional hydrated polymer chains in CY-P-TTSL compared with TTSL which were also functionalized as DSPE-PEG2000, covering the surface of liposomes and prevent them from clearance.52 Furthermore, as shown in table S4, CY-P-TTSL has the highest area under the curve (AUC) during 0~24 h, nearly 15-fold higher than free DOX, and the blood clearance rate (CL) of CY-P-TTSL was calculated to be 0.027 ± 0.015 L/h/kg,


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being the lowest in three groups, 5 times lower than that of free DOX (0.173 ± 0.120 L/h/kg). All these data demonstrated that inclusion of small amount of polymers did not compromise in vivo stability of TTSL. In opposite, it might even get improved to some extent. Moreover, DOX-loaded CY-P-TTSL displayed significantly better blood circulation ability compared with free drug, which is very essential for enhanced tumor accumulation and better drug efficacy.


25


Figure

5.

Pharmacokinetics

study

and

tumor

imaging

Page 26 of 72

of

CY-P-TTSL.

(a)

Pharmacokinetics profiles of CY-P-TTSL, TTSL and free DOX following i.v. at DOX dose of 10 mg/kg. Data are presented as mean ± s.d. (n = 5). (b) BALB/c mice bearing 4T1 tumors were injected with CY-P-TTSL at a DOX dose of 10 mg/kg, and NIR fluorescence images at selected time-points were captured. (c) In vivo time-dependent average fluorescence intensity of tumor. Mean ± s.d. (n = 3). (d) At 24 h post-injection, mice were sacrificed and collected organs were visualized. (e) Normalized Cy7.5 signal in (d). The fluorescent signal of organs was normalized to that in the muscle. Data are presented as mean ± s.d. (n = 3).

2.6 In Vivo Tumor Distribution As a result of prolonged blood circulation time, CY-P-TTSL might also passively accumulate at tumor while circulating in blood. To verify this assumption, in vivo tumor imaging studies were performed. CY-P-TTSL at a DOX concentration of 10 mg/kg was injected intravenously into mice bearing subcutaneous 4T1 breast cancer xenografts and then monitored by IVIS SPECTRUM. The fluorescent signal of Cy7.5 could be


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observed as early as 1 h post-injection and continued to increase over the next day (Figure 5b, c). The highest tumor accumulation was achieved at 24 h and the mice were euthanized then. Next, the excised organs and tumor were imaged as Figure 5d and the fluorescence was normalized to muscle to yield the organ to muscle ratio (Figure 5e). Although the F.I. in tumors was not as strong as livers, they still had near 20-fold fluorescent signal compared with muscles, confirming the accumulation of P-TTSL in tumor area. These results indicate that it is possible to trigger DOX release from CY-PTTSL under HT at both blood vessel and tumor interstitial fluids, making the best use of DOX concentration gradients. 2.7 In Vivo Tumor Penetration The dense interstitial space, extracellular matrix (ECM), and tumor stroma, along with elevated IFP are major physical barriers hindering nanoparticles delivery into tumor tissues.53 For instance, tumor IFP could be in the range of 5–10 mmHg or even higher,9 which can reduce the pressure gradient in interstitial space to near zero, making it much more difficult for nanomedicines like liposomes to transport into deep tumor tissue compared with free drugs.7 Since CY-P-TTSL showed good tumor accumulation and


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long blood circulation ability, it is a good strategy to trigger its DOX release at tumor vessels and interstitial fluids with HT, resulting in deeper tumor penetration via the form of small drug molecules. Here, immunostaining of tumor sections was performed to verify the HT-induced DOX deep penetration. As presented in Figure 6, for CY-P-TTSL group treated with HT, DOX signal was found widely spread around tumor vasculature and distributed deeply into the tissues. In contrast, very weak fluorescence of DOX was observed via normothermia (NT) treatment. And for TTSL, although HT treatment could also induce more DOX infiltration compared with NT, the DOX signal was not as strong as CY-PTTSL. Combining these results together, CY-P-TTSL strongly improved the poor thermo-sensitivity of TTSL when HT was applied. And this quick release of DOX would result in high concentration gradient in tumor vessels and interstitial fluids, leading to enhanced in vivo tumor penetration.54


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Figure 6. In vivo fluorescence distribution of DOX from CY-P-TTSL and TTSL in 4T1 tumor slices with or without HT treatment for 5 min at 24 h post-injection. Cell nuclei were stained with Hoechst 33342 and tumor vessels were labeled with anti-CD31 antibody. Scale bars: 100 μm.

2.8 In Vivo Anti-Tumor Study


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Finally, we treated 4T1-bearing BALB/c mice with CY-P-TTSL and several other formulations via i.v. injection after tumor volume reached 50-100 mm to explore their in

vivo anti-tumor efficacy. 3 As CY-P-TTSL exhibited a maximum tumor accumulation at 24 h during tumor distribution study, thus tumor sites were heated locally for 5 min 24 h post-injection in this experiment, and the real-time temperature was kept at 42 °C when NIR laser was applied, monitored by thermal imaging camera (Figure 7a, b).


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Figure 7. In vivo anti-tumor study of different formulations on 4T1 tumor-bearing mice. (a) Procedure of tumor treatment with liposomes and subsequent local heating. (b) In


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vivo IR thermal imaging of tumor under irradiation of laser. (c) Average volumes of 4T1 tumor after initial treatment with different formulations. For each group, formulation was treated 5 times separately at day 1, 3, 5, 7, 9. Mean ± s.d. (n = 6), **P < 0.01. (d) Survival rates of mice bearing 4T1 tumors after different treatments. *P < 0.05, ***P < 0.001 (e) H&E staining images of tumor sections collected from different treated groups of mice on day 15 post initial treatment. Scale bars: 100 μm.

As depicted in Figure 7c, d, the anti-tumor efficacy between PBS and CY-P-TTSL W/O DOX HT groups showed no significant difference over a time course of 21 days, indicating that HT treatment alone had no effect on inhibition of tumor growth. For TTSL HT and NT groups, although both showed better drug efficacy compared with PBS group, HT treatment did not result in significant improvement in survival time (P = 0.219). In contrast, CY-P-TTSL HT group exhibited significantly longer survival time than CY-PTTSL NT group (P < 0.05). Also, on day 21, the tumor volume of CY-P-TTSL HT group was limited to 331.69 ± 114.31 mm3 (Figure 7c, S11), notably lower than that of TTSL HT group (657.14 ± 114.89 mm3), showing significantly better anti-tumor efficacy. H&E


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staining was also applied to evaluate cellular apoptosis in the tumor sites (Figure 7e). It’s noticed that CY-P-TTSL HT group exhibited the lowest tumor cell density and smaller nucleus shape, demonstrating more tumor apoptosis and necrosis than other groups. To summarize, the anti-tumor study verified that CY-P-TTSL could achieve good therapeutic effect with only 5 min’s maintenance of HT environment. This time is shorter than previous studies,6 which is not only helpful for decreasing damage to normal cells,55 but also a proof for the quick response of CY-TTSL to HT. Correspondingly, the differences between HT/NT treatments and better drug efficacy relative to TTSL for CYP-TTSL might be caused by enhanced tumor penetration of free DOX, supported by the results from in vivo tumor penetration study. Again, these data suggest that with only inserting 1% p(NIPAM-r-HPMA) into TTSL, the thermo-sensitivity could be significantly improved.


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Figure 8. DOX-induced myocardial injury in vivo: (a) H&E staining images of heart sections collected from different treated groups of mice on day 15 post initial treatment. Scale bars: 100 μm (50 μm for zoom in picture). (b) Semi-quantitative analysis of the amount of infiltrated neutrophilic granulocyte nodes in different treated groups, as is indicated by the solid arrow in (a).


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Finally, since it is widely known that free DOX has strong cardiotoxicity,56 the heart toxicity of CY-P-TTSL was studied through H&E staining and the results were showed in Figure 8a, b. A lot of inflammation sites were observed in group treated by free-DOX, indicating intense infiltration with neutrophil granulocytes.57 However, for all the groups treated by liposomal formulation, the total count of inflammation sites was extremely decreased, showing no significant cardiotoxicity, which is consistent with enhanced in

vivo drug circulation ability in the PK study. The histological examination of liver, spleen, lung, and kidney were also applied and the results are shown in Figure S12. No significant change was observed in other groups compared with PBS treatment, further verifying the biocompatibility of polymer-based liposomes.

3. Conclusions

In this study, we successfully developed a novel highly thermo-sensitive polymerbased liposome, using p(NIPAM-r-HPMA), p(HPMA-r-APMA)-Cy7.5 and TTSL. With only including 1% p(NIPAM-r-HPMA) onto lipid bilayer, P-TTSL could achieve extremely quick release of encapsulated DOX under mild HT, while maintaining its stability under


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body temperature. To the best of our knowledge, no previous study achieved this balance with polymer amount as low as we did. Also, much better cellular uptake kinetics and tumor spheroid penetration of free DOX in 4T1 cell lines were observed in P-TTSL compared with TTSL under HT as a result of good thermo-sensitivity. Furthermore, by co-inserting Cy7.5 conjugated p(HPMA-r-APMA) into TTSL, formed CY-P-TTSL showed excellent in vivo imaging ability, along with enhanced blood retention in comparison with free DOX, which ensures reduced cardiotoxicity. Moreover, with 808 nm NIR laser, HT-induced DOX release in tumor sites was successfully achieved, and results in deep tumor penetration, strong tumor apoptosis and growth inhibition in 4T1-bearing BALB/c mice, indicating its potential to be clinically used for cancer treatment.

4. Materials and Methods

4.1 Materials. N-isopropylacrylamide (NIPAM), N-(2-hydroxypropyl) methacrylamide (HPMA), N-(3aminopropyl) methacrylamide (APMA), and 4-Cyano-4-[(dodecylsulfanylthio-carbonyl)


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sulfanyl] pentanoic acid were obtained from Sigma-Aldrich, Inc. (Shanghai, China). 2,2′azobis(2-methyl-propionitrile)

(AIBN),

and

2,2'-Azobis[2-(2-imidazolin-2-yl)propane]

dihydrochloride (VA-044) were purchased from J&K Scientific Ltd. (Beijing, China). Hydrogenated

Soybean

Phospholipids

(HSPC),

1,2-distearoyl-sn-glycero-3-

phosphoethanol-amine-N-[methoxy(poly-ethylene glycol)-2000] (DSPE-PEG2000) were purchased from A.V.T. Pharmaceutical Co., Ltd. (Shanghai， China). 1,2-Dipalmito-ylsn-glycero-3-phosphocholine

(DPPC)

was

purchased

from

Sigma-Aldrich,

Inc.

(Shanghai, China). Cholesterol (Chol) was obtained from J&K Scientific Ltd. (Beijing, China). Doxorubicin hydrochloride was purchased from Dibo biological technology Co., Ltd. (Shanghai, China). Cy7.5 NHS ester was obtained from Lumiprobe Company (Maryland, U.S.A.). 5(6)-TAMRA ethylenediamine was purchased from AAT Bioquest, Inc. (Sunnyvale, CA). Anti-CD31 antibody [MEC 7.46] (ab7388) was purchased from Abcam (Shanghai, China). 4.2 Animals Female BALB/c mice of 18–20 g were obtained from Peking University Health Science Center (Beijing, China), and kept under specific pathogen free (SPF) condition


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with free access to standard food and water. All care and handling of animals were performed with the approval of the Ethics Committee of Peking University. 4.3 Syntheses of p(NIPAM-r-HPMA) and p(HPMA-r-APMA). For the synthesis of p(NIPAM-r-HPMA), the polymerization was carried out by RAFT chemistry,

with

initiator

AIBN

and

chain

transfer

agent

(CTA)

4-Cyano-4-

[(dodecylsulfanyl-thiocarbonyl)-sulfanyl] pentanoic acid (CDSP) at a molar ratio of [CTA]/[Initiator] = 10:1, [monomer]/[CTA] = 100:1. The ratio between NIPAM and HPMA varies according to the experiment needs to get polymers with different LCST. The typical synthesis was carried out as follows: NIPAM (600 mg, 95 eq.), HPMA (40 mg, 5 eq.), AIBN (0.91 mg, 0.1 eq.), and CDSP (22.46 mg, 1 eq.) were dissolved in 2 mL dioxane in a 5 mL ampule. The mixture was degassed by five cycles of freezepump-thaw under nitrogen and vacuum atmosphere, and later stirred at 60 °C for 24 h. After polymerization, the mixture was precipitated into cold diethyl ether. The residue was gained and dissolved in the DMF and next dialyzed in distilled water and lyophilized to obtain a white powder. The polymers were characterized by 400 MHz 1H-NMR, and gel permeation chromatography.


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For the polymerization of p(HPMA-r-APMA), the main procedure was similar as above, while the initiator was changed to VA-044 and the molar ratio of [CTA]/[Initiator] = 3:1, [monomer]/[CTA] = 100:1. The typical synthesis was carried out as follows: HPMA (200 mg, 97 eq.), APMA (7.69 mg, 3 eq.), VA-044 (1.55 mg, 0.33 eq.), and CDSP (4.96 mg, 1 eq.) were dissolved in 2 mL CH3OH in a 5 mL ampule. The mixture was degassed by five cycles of freeze-pump-thaw under nitrogen and vacuum atmosphere, and later stirred at 50 °C for 24 h. After polymerization, the mixture was precipitated into cold acetone. The residue was gained and dissolved in the water and next dialyzed in distilled water and lyophilized to obtain a white powder. The polymers were characterized by 400 MHz 1H-NMR, and fast protein liquid chromatography. 4.4 Conjugation of Fluorescent Probes to Polymers. In the preparation of TMR-p(NIPAM-r-HPMA), TMR was conjugated with the terminal carboxylic group of polymer. Briefly, the polymer was mixed with DCC and NHS at a molar ratio of 1:1.2:1.4 in CH3OH overnight. Next, TAMRA ethylenediamine (1 eq.), TEA (3 eq.) was added and further reacted for 24h. Cy7.5 NHS ester was conjugated to p(HPMA-r-APMA) with the amino group on APMA block. Briefly, the molar ratio of total


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amino groups on polymer and Cy7.5 NHS ester was 1.2:1. The polymer and Cy7.5 NHS ester were mixed together in CH3OH and reacted for 24 h. After reaction, both probeconjugated polymers were purified by ultrafiltration (100 kDa MWCO, Millipore). 4.5 Preparation of DOX-loaded Liposomes. Liposomes were prepared by the lipid film hydration and extrusion method.24 First, lipids were dissolved in the chloroform. Next, the organic solvent was evaporated to generate a dry film and flushed with N2 stream to remove residual chloroform. Ammonium sulfate buffer (240 mM (NH4)2SO4) was used to hydrate the lipid film at 60 °C. To produce small single unilamellar liposomes, the size was reduced by extrusion though 100 nm polycarbonate filters 10 times using a Mini-Extruder (Avanti Polar Lipids, Inc., AL, U.S.A.). The formulation was DPPC/HSPC/Chol/DSPE-PEG2000 at a molar fraction of 50:25:15:3. For DOX loading, liposome solutions were first passed through Sephadex G-50 column to generate ammonium sulfate gradients and DOX was next added at a mass ratio of 5%, incubated at 37 °C for 5 h. Afterwards, liposomes were passed through Sephadex G-50 column to remove any free DOX.


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To get P-TTSL, DOX-loaded liposomes were incubated with p(NIPAM-r-HPMA) and p(HPMA-APMA) at a given mass ratio at 25 °C for 1 h. After post-insertion, the unattached copolymers were removed through ultrafiltration (3000 rpm, 1 h). 4.6 Characterization of P-TTSL. The hydrodynamic diameter, PDI and zeta potential of different liposomes were measured at 25 °C (if not other mentioned) by dynamic light scattering (DLS) using a Zetasizer Nano series Nano-ZS (Malvern. U.K). The morphology and structure of the liposomes were analyzed by JEM1400PLUS transmission electron microscope (JEOL, Inc., USA); UV−Vis spectrometer (UH5300, Hitachi Co., Japan) and Fluorescence Spectrophotometer (F-7000, Hitachi Co., Japan) were used to study the absorption and fluorescent spectra of prepared liposomes. The phase transition temperature of formed liposomes was evaluated by Q2000 differential scanning calorimetry (DSC, TA instruments, USA); The transmittance of different liposomes at varying temperatures was detected by FlexStation 3 multi-mode microplate reader (Molecular Devices, LLC). The post-insertion efficiency of polymer to lipid bilayer was characterized as follows: After incubation of TMR-p(NIPAM-r-HPMA) and liposome solutions (W/O DOX) at given


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mass ratio and time period, the mixture was purified by ultrafiltration (100 kDa, 3000 rpm). The concentration of TMR-p(NIPAM-r-HPMA) in filtrate was determined by UV−Vis spectrometer through the peak absorbance of TMR at 550 nm, according to a standard curve, and the mass (Mfiltrate) could be calculated. Next, with the mass of total polymer before post-insertion (Mtotal), the percentage of inserted polymer could be described as: Inserting efficiency (%) =

Mtotal ― Mfiltrate × 100% Mtotal

(1)

The remaining percentage of DOX after post-insertion was analyzed by similar procedures: after incubation of TMR-p(NIPAM-r-HPMA) and liposome solutions at given mass ratio for 1 h. Solution (1 mL) was taken out to measure the absorbance of DOX at 500 nm (Abefore). Then, this solution was ultra-filtered with 10 mL PBS. After ultrafiltration, the solution in upper layer was collected and replenished to 1 mL, and the absorbance of DOX was measured again (Aafter). Therefore, the percentage of DOX remaining in the liposome could be defined as: DOX remaining (%) = Aafter Abefore × 100%

(2)


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4.7 In Vitro Drug Release and Stability The drug release experiment was carried out in PBS 7.4 at different temperatures according to the methods used in published papers.11, 18, 58 First, the PBS solution was preheated to the target temperature, then calculated amount of liposome solution was added to make the concentration of DOX diluted to 10 μg/mL. Next, 300 μl solution was taken out and cooled inside the ice bath immediately at each time point. After the solution returned to room temperature, fluorescence intensity of DOX was measured in the spectrophotometer at 485 nm excitation wavelength and 592 nm emission wavelength. And the percentage of DOX released at each time point was determined according to the following equation: Ft ― F0

DOX released (%) = FTX ― F0 × 100%

(3)

Where F0 is the background fluorescence intensity of DOX-loaded liposome at a concentration of 10 μg/mL in PBS 7.4, Ft is the fluorescence intensity of collected samples at different time points, and FTX refers to the fluorescence intensity after


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diluting DOX-loaded liposome to a concentration of 10 μg/mL in 1% Triton X-100 in PBS 7.4. For IR-tuned DOX release, solutions containing CY-P-TTSL were irradiated to different temperature by 808 nm laser (Changchun Laser Optoelectronics Technology Co., Ltd., China). And the release percentage was determined by the same method as above. For in vitro 4 °C storage stability, DOX-loaded liposomes were stored at 4 °C in PBS 7.4 solution, and measured at different time points. The in vitro serum stability was measured in 10% fetal bovine serum (FBS) in PBS 7.4 by monitoring the fluorescence intensity of DOX at 37 °C for different incubation time periods. 4.8 In Vitro Photothermal Effect. PBS solution, free DOX, P-TTSL and CY-P-TTSL at a final Cy7.5 concentration of 6.66 μg/mL were put into 1 mL glass bottle, and irradiated by the 808 nm laser with a power density of 500 mW/cm2 respectively until the temperature become nearly unchanged. Real-time imaging was monitored and infrared thermographic maps were obtained by the infrared thermal imaging camera (Fotric 226, FOTRIC., China).


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4.9 In Vitro Cytotoxicity. MTT assay was used to evaluate the cellular toxicity of different formulations. 4T1 cells were seeded into a 96-well plate and incubated under 37 °C with 5% CO2 for 24 h. Then, the supernatants of plate were discarded and free DOX, TTSL and P-TTSL were added at a DOX concentration of 0.01, 0.1, 1, 5, 10, 100 μg/mL in complete RPMI 1640. For the cytotoxicity at 37 °C, the plates were further incubated at 37 °C with 5% CO2 for 4 h. For the cytotoxicity at 42 °C, the plates were heated by water bath at 42 °C for 5 min and next cooled before incubated at 37 °C with 5% CO2 for 4 h. After incubation, the supernatants of plates were discarded and washed 3 times with PBS before replaced by RPMI 1640 and subsequently incubated at 37 °C overnight. Then, the solution was displaced by 100 μL MTT (0.5 mg/mL) and incubated for 4 h. Next, the medium was removed and 200 μL of DMSO was added to evaluate the cytotoxicity according to the absorbance of each well at 560 nm with a microplate reader (Multiskan FC, Thermo). 4.10 In Vitro Cellular uptake.


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For quantitative analysis, 4T1 cells were incubated with TTSL and P-TTSL at 5 μg/mL DOX

in RPMI 1640 for 5 min at 37°C and 42 °C, respectively. The cells were

subsequently incubated with liposomes under 37 °C for 4 h before washed by PBS 7.4 for 3 times. Afterwards, cells were trypsinized and collected for measurement of the intrinsic fluorescence of DOX by flow cytometer (Cytoflex, Beckman Coulter, Inc). Confocal laser scanning microscope (CLSM, TCS-SP8, Leica, Germany) was used to observe the time-dependent cellular uptake of DOX.41,

59-60

Briefly, cells grown on

confocal glass bottom dish were cultured with TTSL and P-TTSL at a DOX concentration of 2 μg/mL. Then they were incubated at 37 °C and 42 °C respectively for different time periods before washed with PBS and fixed with 4% paraformaldehyde. Afterwards, Hoechst 33342 was added to replace the medium and incubated for 30 min, followed by washing 3 times with PBS before image capture using CLSM. 4.11 In Vitro Penetration of Tumor Spheroid. To prepare the three-dimensional tumor spheroids, 4T1 cells were seeded at a density of 5 × 104 cells/mL per well in 48-well plates (20 μL/plate). Several days after, when the spheroids were recognizable, they were transferred to the plates which were


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coated by 200 μL of 2% low-melting-temperature agarose and further incubated to reach the size of 200 ~500 μm. TTSL and P-TTSL were diluted to a DOX concentration at 5 μg/mL and added to confocal glass bottom dish with the tumor spheroids. Next they were heated at 42 °C for 2 min and then incubated at 37 °C for given time periods before image capture by CLSM. 4.12 In Vivo Pharmacokinetic Profiles. The experiment was performed according to the reported method.61 Female BALB/c mice of different groups were i.v. administrated with different formulations at an equivalent DOX of 10 mg/kg through the tail vein. Blood samples (50 μL) were collected for each mouse at the time points of 0.033, 0.25, 0.5, 1, 3, 6, 12 and 24 h after intravenous injection. Next, the samples were centrifuged at 2000 rpm for 15 min to remove the plasma. Then 10 μL of plasma was mixed with 200 μL of acidified methanol. Afterwards the resulted mixture was fully vortexed for 2 min to extract the DOX, followed by centrifugation at 10000 rpm for 15 min. The supernatant was collected and the DOX fluorescence was measured by spectrophotometer (λex/em = 485 nm/592 nm). By comparing the fluorescence with a calibration curve generated from known amounts of


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DOX in mouse plasma: F.I.= 4667.8×C + 96.47, R= 0.9993 (Figure S10), the DOX concentration at different time points could be determined. The serum volume of mice was estimated to be around 60 mL/kg body weight to determine the initial serum concentration.62 4.13 In Vivo Tumor Distribution Study. Female BALB/c mice were anesthetized and then 50 μL of PBS containing 6 × 105 4T1 cells was subcutaneously injected into the right flank. Two weeks later, when the tumor volume reached around 200 mm3, each mouse was i.v. administrated with CY-PTTSL at a DOX of 10 mg/kg via the tail vein. Then the whole body fluorescent distribution was observed by an in vivo imaging system (IVIS SPECTRUM, Perkinelmer, U.S.A.) at different time points after injection. Then the mice were sacrificed, and their organs were subjected to ex vivo fluorescence imaging. In vivo imaging conditions at λem = 840 nm, λex = 745 nm was used to acquire the NIR signals of Cy7.5. 4.14 In Vivo Tumor Penetration. Immunofluorescence labeling was applied to evaluate the drug penetration at tumor sites in vivo according to the methods from published literature.63 Briefly, two groups of


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4T1 tumor-bearing mice were i.v. administrated with TTSL and CY-P-TTSL respectively at an equivalent DOX concentration of 5 mg/kg via the tail vein. 24 h after i.v. injection, the tumor site of mice was irradiated by 808 nm NIR laser for 5 min in CY-P-TTSL group. The infrared thermal imaging camera was used to monitor real-time temperature of tumor and keep it at 42 °C by changing the laser power. For TTSL, the tumors were heated by water bath for 5 min at 42 °C. 24 h post NIR irradiation, the mice were euthanized, followed by snap frozen of their tumors and being cut into 10 μm sections. The slices were fixed in 4% paraformaldehyde and rinsed with PBS three times, then blocked with 10% BSA for 1 h at room temperature. Subsequently, the slices were incubated with rat anti-mouse CD31 antibody at 4 °C overnight. Later, FITC-conjugated secondary antibody was added to stain the slices at 37 °C for 2 h. The slides were mounted with Hoechst 33342-containing medium and further imaged by confocal microscopy. 4.15 In Vivo Antitumor Efficacy. Female BALB/c mice were anesthetized and then 50 μL of PBS containing 6 × 105 4T1 cells was slowly injected into the right upper thigh subcutaneous region. Several


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days after, when the tumor volume reached around 50 mm3, mice from different groups was i.v. administrated with different formulations at an equivalent DOX concentration of 5 mg/kg (except CY-P-TTSL W/O DOX group) through the tail vein. Specifically, DOX was not encapsulated into liposomes as for CY-P-TTSL W/O DOX group, and the total inject dose was equivalent to other groups according to the amount of lipid. For each group, formulation was given 5 times separately at day 1, 3, 5, 7, 9. For the CY-P-TTSL HT groups, they were irradiated for 5 min 24 h post-injection by 808 nm laser. The infrared thermal imaging camera was used to monitor real-time temperature of tumor and keep it at 42 °C by changing the laser power. HT treatment of TTSL group was carried out in water bath under 42 °C for 5 min. Tumor volume and weight were measured every other day after initial treatment. When tumor size reached 1400 mm3 (Height×Width2× 0.5), the mice were euthanized. 4.16 Biosafety Evaluations. Healthy Female BALB/c mice were administrated with different formulations according to the same experimental methods described in antitumor efficacy study. After 15 days, several mice were sacrificed and their major organs including liver, heart, kidney,


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spleen, and lung along with tumors were collected for investigating the histopathology changes based on H&E staining. 4.17 Statistical Analysis. The data shown here were presented as mean ± standard deviation (s.d.). Significant differences were evaluated using an independent-samples t-test or Analysis of Variance (ANOVA) depending on the amount of treatment groups and variables. For survival analysis, it was calculated by the log-rank test.

ASSOCIATED CONTENT

Supporting Information. Synthetic routes and characterizations of p(NIPAM-r-HPMA) and p(HPMA-r-APMA); In vitro cellular uptake of DOX by 4T1 cells at 37 °C and semiquantitative analysis; Tumor spheroids incubated with formulations for 1 h; Characterizations of CY-P-TTSL; Calculated PK parameters; H&E staining images of liver, spleen, lung, and kidney sections. The following files are available free of charge. brief description (file type, i.e., PDF)


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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] Tel.: +86-10-82805084.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was financially supported by the National Key Research and Development Program of China (2017YFA0205600 and 2016YFA0201400) and the National Natural Science Foundation of China (NSFC) grants (81473157, 81622046, and 81671817). Y.W. is grateful to the startup funding from the Thousand Young Talents program of China.


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(2) Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C., Cancer nanomedicine: progress, challenges and opportunities. Nat Rev Cancer 2017, 17 (1), 20-37, DOI: 10.1038/nrc.2016.108.

(3) Leserman, L. D.; Barbet, J.; Kourilsky, F.; Weinstein, J. N., Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A. Nature 1980, 288 (5791), 602-4.

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For Table of Contents Use Only

Quick-Responsive

Polymer-Based

Thermo-Sensitive

Liposomes for Controlled Doxorubicin Release and Chemotherapy

Yulin Mo, Hongliang Du, Binlong Chen, Dechun Liu, Qingqing Yin, Yue Yan, Zenghui Wang, Fangjie Wan, Tong Qi, Yaoqi Wang, Qiang Zhang, Yiguang Wang*


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Quick-Responsive Polymer-Based Thermosensitive Liposomes for

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