Dual-responsive nanoparticles with transformable shape and reversible charge for amplified chemo-photodynamic therapy of breast cancer

Herein, we designed a dual-response shape transformation and charge reversal strategy with chemo-photodynamic therapy to improve the blood circulation time, tumor penetration and retention, which finally enhanced the anti-tumor effect. In the system, hydrophobic photosensitizer chlorin e6 (Ce6), hydrophilic chemotherapeutic drug berberrubine (BBR) and matrix metalloproteinase-2 (MMP-2) response peptide (PLGVRKLVFF) were coupled by linkers to form a linear triblock molecule BBR-PLGVRKLVFF-Ce6 (BPC), which can self-assemble into nanoparticles. Then, positively charged BPC and polyethylene glycol-histidine (PEG-His) were mixed to form PEG-His@BPC with negative surface charge and long blood circulation time. Due to the acidic tumor microenvironment, the PEG shell was detached from PEG-His@BPC attributing to protonation of the histidine, which achieved charge reversal, size reduction and enhanced tumor penetration. At the same time, enzyme cutting site was exposed, and the spherical nanoparticles could transform into nanofibers following the enzymolysis by MMP-2, while BBR was released to kill tumors by inducing apoptosis. Compared with original nanoparticles, the nanofibers with photosensitizer Ce6 retained within tumor site for a longer time. Collectively, we provided a good example to fully use the intrinsic properties of different drugs and linkers to construct tumor microenvironment-responsive charge reversal and shape transformable nanoparticles with synergistic antitumor effect.

MMP-2, while BBR was released to kill tumors by inducing apoptosis. Compared with original nanoparticles, the nanofibers with photosensitizer Ce6 retained within tumor site for a longer time. Collectively, we provided a good example to fully use the intrinsic properties of different drugs and linkers to construct tumor microenvironment-responsive charge reversal and shape transformable nanoparticles with synergistic antitumor effect.

Introduction
In recent years, although nanoparticles are widely used in tumor therapy 1 , the tumor heterogeneity and complex tumor microenvironment seriously affect the distribution, penetration and retention of nanoparticles, attenuating antitumor effect 2e4 . The clearance by mononuclear phagocyte system (MPS) in the blood circulation makes it difficult for nanoparticles to enrich in the tumor site, the high interstitial fluid pressure in the tumor parenchyma tends to pump the nanoparticles back to the blood, and the dense matrix and high solid stress hinder the penetration of nanoparticles into deep tumors 5 . Therefore, it is important to overcome these barriers by designing novel nanoparticles, which has attracted increasing attention in the field 6e8 .
Physical properties of nanoparticles, such as size 9 , shape 10 , and surface characteristics 11,12 , play an important role in the tissue distribution, tumor penetration and retention. Generally, spherical nanoparticles have good tumor penetration, while nanofibers have good tumor retention 13 . Therefore, shape transformable nanoparticles are designed to overcome the shape controversial and improve intratumor drug delivery 14,15 . Matrix metalloproteinase-2 (MMP-2) is expressed excessively in breast cancer 16,17 , which could specifically cleave the peptide PLGVR 18,19 . To enable MMP-2-responsive shape transformation, we designed a new peptide PLGVRKLVFF. In the peptide, KLVFF is derived from Ab peptide, which can form b-sheet and finally construct nanofibers 20 . However, the proline (P) in the peptide can effectively inhibit b-sheet and thus inhibit the formation of nanofibers 21 . After introducing the hydrophobic head and hydrophilic tail into the PLGVRKLVFF peptide, the resulted linear molecule can form spherical nanoparticles due to the hydrophobic force, and change to nanofibers under MMP-2 hydrolysis, improving the tumor retention of hydrophobic drug.
Carrier-free nanoparticles have very high drug loading capacity and could avoid materials-originated side effect, which have been the most promising strategy in the drug delivery 22 . Therefore, we proposed to select suitable drugs for shape transformable nanoparticle construction. Chlorin e6 (Ce6) is a widely used hydrophobic photosensitizer for photodynamic therapy (PDT) 23 . Berberrubine (BBR) is a hydrophilic and positively charged antitumor drug that can achieve anti-tumor effect through a variety of mechanisms, such as promoting apoptosis, and producing reactive oxygen species (ROS) 24 . So, the Ce6 and BBR were conjugated by PLGVRKLVFF peptide (BBR-PLGVRKLVFF-Ce6, BPC) to construct a linear molecule that can form spherical nanoparticles due to the hydrophobic force. The MMP-2responsive shape transformable BPC nanoparticles showed positive surface charge, which could penetrate deep tumor by caveolae-mediated endocytosis and exocytosis 25e27 .
Although the shape transformation strategy can effectively improve the retention and penetration of nanoparticles at the tumor site, positively charged nanoparticles tend to be cleared by MPS in blood circulation, which attenuating the tumor targeting delivery efficiency 28 . Therefore, charge reversal has become an effective strategy to improve blood circulation and tumor distribution. Because of the precise location, real-time monitoring, microenvironment response, and low toxicity, stimuli-responsive nano-based drug delivery systems have been a promising approach in treatments 29,30 . Due to the acidic tumor microenvironment, pH response become one of the common trigger conditions. To provide the BPC nanoparticles with pH-responsive charge reversal property, polyethylene glycol-histidine (PEG-His) was used to coat the BPC to form a negatively charged nanoparticle (PEG-His@BPC) due to the electrostatic adsorption. The negative charge of PEG-His@BPC make it have long blood circulation time. After reaching the tumor site, the histidine was protonated, and PEG was detached to expose the positively charged BPC for tumor penetration, and MMP-2-triggered shape transformation.
In this study, we designed a pH-responsive charge reversal and MMP-2-responsive shape transformable nanoparticle PEG-His@BPC. The spherical PEG-His@BPC with negative charge had long blood circulation time and could passively target to tumor tissue. Due to the protonation of histidine in acidic tumor microenvironment, PEG shell fell off, and the positively charged BPC was exposed with good tumor penetration. Simultaneously, the highly expressed MMP-2 cleaved the PLGVR fragment, and the external hydrophilic BBR was detached and released to promote tumor cell apoptosis. At the same time, due to the switch of hydrophilic-hydrophobic ratio and the b-folding formation, the remaining pep-Ce6 was reassembled into nanofibers, thus increasing the tumor retention and cellular uptake. Under laser irradiation, high levels of ROS induced by Ce6 could further induce tumor cell apoptosis, which has synergistic effect with BBR. Based on this dual-responsive shape transformation combination therapy, self-delivered PEG-His@BPC achieved enhanced chemo-photodynamic combinational therapy against breast cancer (see Scheme 1).

Materials
Fmoc-PLGVRK(Dde)LVFF (MW Z 1561) was purchased from DECHI Biotechnology Co., Ltd. (Shanghai, China). Ce6 was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). BBR was purchased from PUFEIDE Biotechnology Co., Ltd. (Chengdu, China). CDM and t-butyloxy carbonyl (Boc)ethylenediamine were purchased from McLean Biochemical Technology Co., Ltd. (Shanghai, China). Mouse 4T1 cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in complete RPMI-1640 cell medium (YUANPEI, Shanghai, China) containing 10% fetal bovine serum, 100 U/mL penicillin G and 100 U/mL streptomycin sulfate. The cells were cultured at 37 C and 5% CO 2 . Female BALB/c mice (5e6 weeks, 18e22 g) and female BALB/c nude mice (4e5 weeks, 18 AE 2 g) were purchased from ENSI-WEIER Co., Ltd. (Chongqing, China). All animal experiments were conducted under the guidelines evaluated and approved by the Ethics Committee of Sichuan University.

Synthesis of Ce6-NHNH 2
Ce6 (3 mg) was dissolved in DMF (2 mL), then EDC$HCl (2.9 mg) and NHS (1.74 mg) were added to activate the carboxyl group. Then Boc-ethylenediamine (0.89 mg) was dissolved in DMF (1 mL) and mixed with Ce6. The mixture was stirred in darkness at room temperature (RT) for 12 h to obtain Boc-NHNH-Ce6. It was diluted to 6 mL with ultra-pure (UP) water, HCl was added to adjust pH to 6, and the solution was extracted 5 times with DCM. Then organic phase was merged, and the organic phase was washed with saturated NaCl solution to remove excess water-soluble impurities. DCM was removed by rotary evaporator, DCM and TFA (7:3) were added to remove the Boc under RT for 3 h. The DCM was removed by rotary evaporator again, and the salt of trifluoroacetic acid was obtained. Then saturated NaHCO 3 solution was added to adjust pH to 8. After being extracted with DCM for 5 times, the organic phase was washed with saturated NaCl solution, and the organic phase was concentrated to obtain Ce6-NHNH 2 .

Synthesis of PLGVRK(Dde)LVFF-Ce6
The Fmoc-PLGVRK(Dde)LVFF peptide (5 mg) was dissolved in 2 mL of DMF, and subsequently EDC$HCl (1.84 mg) and NHS (1.1 mg) were added to activate the carboxyl group. The solvent of Ce6-NHNH 2 solution was removed and 2 mL of DMF was added to dissolve Ce6-NHNH 2 , and the solution was put into the activated peptide drop by drop. The mixture was stirred at RT and dark for 12 h. Then 20% 4-methylpiperidine was added to remove the Fmoc, and the mixture was dialyzed for 48 h. The dialysate was freeze-dried and finally PLGVRK(Dde)LVFF was obtained.
Scheme 1 Scheme illustration of the composition of PEG-His@BPC and its therapeutic effect on breast cancer.

Synthesis of BBR-CDM
CDM (1.72 mg) was dissolved in 2 mL of anhydrous DCM at 0 C, and oxalyl chloride (1.22 mg) and anhydrous DMF (1 mL) were added. The mixture was stirred in ice bath for 10 min and then stirred at RT for 1 h. Impurities such as DCM and excess oxalyl chloride were removed by rotary evaporation to get pure acyl chloride. BBR (3 mg), the above acyl chloride and anhydrous pyridine (2 mL) were mixed into anhydrous acetonitrile, and then the mixture was stirred at RT for 3 h to obtain BBR-CDM.

Synthesis of BPC and PEG-His
The PLGVRK(Dde)LVFF and BBR-CDM were dissolved in anhydrous DMF. Then 4-dimethylaminopyridine was added into the solution and the solution was stirred in darkness at RT for 12 h. Hydrazine hydrate solution (2%) was added to remove Dde, and then the BPC was purified by dialysis for 48 h. The mPEG-CM was dissolved in UP water, and EDC$HCl and NHS were added to activate carboxyl group for 3 h. Histidine was added into the mixture and the mixture was stirred for 24 h. Finally, PEG-His was purified by dialysis.

Preparation of PEG-His@BPC micelles
BPC was dissolved in chloroform:methanol (4:1) solvent, then 10fold UP water volume was added. The mixed solvent was emulsified in ice water bath by ultrasonic emulsification (100 W, 5 min). Then organic solvent was removed by rotary evaporator, and the particle size of BPC micelles was measured by Malvern particle size analyzer (Malvern, Nano-ZS90, UK). Finally, the BPC micelles and PEG-His were incubated at RT for 12 h.

MMP-2-responsiveness
One milliliter of 0.5 mg/mL PEG-His@BPC was co-incubated with 300 ng/mL MMP-2 under pH 6.5 or 7.4, and groups without MMP-2 were used as control. The changes of particle size were monitored by Malvern particle size analyzer at 2, 4, 8 and 12 h.

Intracellular transformation
After discarding the culture medium, 2.5% glutaraldehyde fixation solution was added to the cultured 4T1 cells. Cells were fixed at RT for 5 min and then scraped off gently in one direction with cell scraper. The cell fluid was added to the centrifuge tube with Pasteur pipette and then centrifuged for 5 min. The supernatant fluid was discarded, and the fresh 2.5% glutaraldehyde fixed solution was added. The cell mass was gently picked up and suspended in the fixed solution. It was fixed away from light at RT for 30 min and then transferred to 4 C for preservation. Finally, the fixed cells were made into ultra-thin sections and observed by TEM (JEOL, JEM-2100 Plus, Japan).

Penetration and retention in MSC
The low melting point agarose (2% w/v) was dissolved in the cell culture medium, and the solution was added to the 96-well plate (80 mL per well). After the gel was cooled and solidified, 4T1 cells were inoculated on the gel (3 Â 10 3 cells per well) to formed MSC. The MSCs around 500 mm were selected and treated with PEG-His@BPC and PEG-His@BRC. After 12 h, MSCs were washed and fixed. Then the fluorescent intensity was observed by CLSM (ZEISS, LSM 800, Germany). Meanwhile, the medium was replaced with fresh culture medium and incubated for another 12 h. Then MSCs were washed and fixed, and the fluorescent intensity was observed to investigate the tumor retention of nanomedicines 31 .

Calcein-AM/PI double staining
4T1 cells were inoculated on the 24-well plate. After being treated with different formulations for 12 h, 4T1 cells were washed and stained with Calcein-AM (5 mg/mL) and PI (10 mg/mL) for 30 min. Finally, 4T1 cells were washed to remove excess dyes and fluorescent intensity was observed by fluorescence microscope (Nikon, Ts2RFL, Japan).

Biodistribution
4T1 cells (4 Â 10 5 ) were subcutaneously injected into the left mammary fat pads of female BALB/c nude mice. After 7 days, mice with tumor around 100 mm 3 were selected and randomly divided into 4 groups (n Z 3), which were intravenously administrated with PEG-His@BPC, PEG-Phe@BPC, PEG-His@BRC and PEG-Phe@BRC, respectively. The in vivo fluorescence imaging was carried out at 4, 12, 24, 36 and 48 h postintravenous administration by using the Lumina III imaging system (PerkinElmer, IVIS Lumina Series III, USA). All mice were sacrificed after the last imaging, and the major organs and tumors were cleaned, fixed and collected for ex vivo fluorescence imaging. All tumors and organs were dehydrated and frozenly sectioned, then imaged by CLSM (ZEISS). The scanning parameters of fluorescence imaging were as follows: excitation wavelength Z 660 nm, emission wavelength Z 710 nm, visual field Z 12.5 cm, and fluency Z 2 mW/cm 2 . The camera is set to maximum gain and the split-box factor is 4, unless otherwise stated, the same below.

Retention in tumor
BALB/c nude mice 4T1 breast cancer model was constructed as above. One week later, mice with tumor around 100 mm 3 were selected and randomly divided into 4 groups (n Z 3), which were intratumorally injected with PEG-His@BPC, PEG-Phe@BPC, PEG-His@BRC, PEG-Phe@BRC, respectively. The in vivo fluorescence imaging was detected at 0, 8, 12, 24 and 36 h after intratumoral injection. Then the ex vivo fluorescence imaging and fluorescent slices were made as before. In addition, tumors were made into ultra-thin sections and observed by TEM (JEOL).

Anti-tumor effect in vivo
BALB/c mice 4T1 breast cancer models were constructed as above. Six days later, mice with tumor around 80 mm 3 were selected and randomly divided into 9 groups (n Z 6). Mice in different groups were intravenously injected with PBS, BBR, PEG-His@BPC, Ce6þL, PEG-His@BPC þ L, PEG-Phe@BPC þ L, PEG-His@BRC þ L, PEG-Phe@BRC þ L, PEG-His@APC þ L (5 mg/kg Ce6 for each group), respectively.
The 650 nm laser irradiation was carried out at 24 h postintravenous administration. The mice were treated every 3 days for 4 cycles, and the tumor volume and body weight were evaluated and recorded every 2 days from Day 6. All mice were sacrificed on Day 22 post-implantation of tumor, and major organs and tumors were cleaned, fixed and collected. The tumors were weighed and imaged in natural light. The sections prepared from tumors and organs were stained with H&E, Ki67 and TUNEL.

Preparation and characterization of PEG-His@BPC
Mass spectrum showed that there were peaks at 639.0 and 320.0 (Supporting Information Fig. S1A), indicating the successful synthesis of Ce6eNHNH 2 . The peak of m/z Z 487.8 indicated the successful synthesis of BBR-2-(2-carboxyethyl)-3-methylmaleic anhydride (BBR-CDM, Fig. S1B). The peaks at 1796.2 and 2284.0 belonged to PLGVRKLVFF-Ce6 and BPC as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, SHIMADZU, AXIMA-TOF, Japan), indicating the successful synthesis of BPC ( Fig. 1A and B). The peaks at 5000.0 and 5145.4 belonged to PEG-CM and PEG-His, and the peak displacement of 150 indicated the successful synthesis of PEG-His ( Fig. S1C and S1D). The average sizes of BPC micelles and PEG-His@BPC were 55.6 AE 4.3 and 76.7 AE 5.2 nm, respectively ( Fig. 1C and D). When pH reduced from 7.4 to 6.5, the zeta potential of PEG-His@BPC converted from À13.5 AE 2.3 to 18.9 AE 1.7 mV (Fig. 1E), indicating the PEG-His could successfully detach from the particles at acidic condition.

MMP-2-responsiveness
PLGVR is not only a specific responsive peptide sequence of MMP-2 32 , but also has the proline (P) which can effectively inhibit b-sheet and thus inhibit the formation of nanofibers. KLVFF, derived from Ab peptide, often relies on a wide range of intermolecular hydrogen bonds to form b-sheet, which is easy to form nanofibers 20 . Therefore, the change of hydrophilic and hydrophobic force and release of proline inhibition caused by enzymatic hydrolysis can be used to re-form the b-sheet to realize the shape transformation from micelle to nanofiber. MALDI-TOF-MS (SHIMADZU) showed that the peak of PLGVRKLVFF changed obviously after MMP-2 cleavage, and the appearance of multiple peaks may be due to the results of multiple cleavage sites (Supporting Information Fig. S2A and S2B). On this basis, we co-incubated PEG-His@BPC with MMP-2 (denoted as þ E) under pH 6.5 or 7.4, and groups without MMP-2 were used as control. The dynamic light scattering (DLS, Malvern) particle size of PEG-His@BPC þ E at pH 6.5 changed most rapidly and strongly, which reached 2000 nm after 4-h incubation (Fig. 1F). This result indicated the nanoparticles could transform into nanofibers within 4 h. Moreover, transmission electron microscope (TEM, JEOL) showed that PEG-His@BPC had different shapes under different pH and MMP-2 conditions, and a large number of nanofibers appeared only in the presence of MMP-2 at pH 6.5 (Fig. 1G), indicating the PEG-His@BPC could successfully transform from spherical particles to nanofibers in the presence of MMP-2 and acidic condition 21,33,34 . In addition, DLS (Malvern) and TEM (JEOL) showed that enzymolysis of PEG-His@BPC by MMP-2 was slowed down under pH 7.4, but PEG-His@BPC could be quickly cleaved by MMP-2 and transformed into nanofibers under pH 6.5, which may be caused by the shedding of PEG shell at neutral condition.

Cellular uptake
Compared with PEG-His@BPC without MMP-2, the presence of MMP-2 obviously enhanced cellular uptake of PEG-His@BPC under pH 6.5 or 7.4 (1.67 and 1.35 times, respectively), according to both flow cytometry (Agilent, NovoCyte, USA, Fig. 2A) and confocal laser scanning microscopy (CLSM, ZEISS, Fig. 2B). The results demonstrated that the shape transformation can effectively promote the cellular uptake of nanoparticles, which was consistent with the previous reports 14 . Soft nanofibers are more likely to attach to the cell surface as they pass through the cell, rather than external cutting through it like spherical nanoparticles, so the difference in uptake capacity between nanofibers and micelles may be attributed to their stiffness 35 .

Intracellular shape transformation
First, we constructed a control peptide RPGFGNKLVFF that could not be cleaved by MMP-2, which displayed the same peaks before and after it incubated with MMP-2 as determined by MALDI-TOF-MS (SHIMADZU, Fig. S2A). Then we used the peptide to synthesize control nanoparticles PEG-His@BBR-RPGFGNKLVFF-Ce6 (PEG-His@BRC), which could not transform to nanofibers. Finally, we observed the shape transformation of PEG-His@BPC and PEG-His@BRC in 4T1 cells. As we expected, the results showed there were no nanofibers in cells treated with PEG-His@BRC, but plenty of spherical nanoparticles around 50 nm (blue arrow) were found in endosomes or cytoplasm (Supporting Information Fig. S3A). Importantly, the nanofibers (red arrow) were abundant in the cytoplasm of cells treated with PEG-His@BPC (Fig. S3B), indicating that PEG-His@BPC could transform from micelles to nanofibers at the cellular level, which was consistent with previous reports 36 .

Penetration and retention in multicellular spheroids
The tumor penetration and retention of nanomedicines were confirmed by confocal imaging of multicellular spheroids (MSCs). First, the MSCs were treated with PEG-His@BRC and PEG-His@BPC for 12 h. There were similar Ce6 signals in PEG-His@BRC-and PEG-His@BPC-treated MSCs, suggesting that PEG-His@BPC displayed the same excellent penetration capacity as non-transformable spherical nanoparticles (Supporting Information Fig. S4A and S4B). Then each group of nanomedicines was replaced with fresh culture medium and incubated for another 12 h to investigate the tumor retention of nanomedicines. The results showed PEG-His@BRC was excreted rapidly, which tended to vanish at 80 mm. In contrast, there were strong Ce6 signals retained in the MSCs treated with PEG-His@BPC (Fig. 2C). The fluorescence signals of PEG-His@BPC group were 1.87-fold higher than that of PEG-His@BRC group at 120 mm of depth, indicating PEG-His@BPC displayed better tumor retention than nontransformable PEG-His@BRC (Fig. 2D). Meanwhile, the difference between MSCs treated with PEG-His@BRC and PEG-Phe@BRC was compared to confirm the contribution of charge reverse in spheroids penetration ( Fig. S4C and S4D). The results showed that fluorescence intensity of PEG-His@BRC was 1.33fold higher than that of PEG-Phe@BRC at 120 mm of depth, which proved that the charge reversal was beneficial to the tumor penetration. A good tumor penetration achieved by charge reversal could enrich more nanoparticles in the deep part of the tumor, which could cooperate with the enhancement of tumor retention caused by shape transformation to enhance the delivery efficiency. Solid tumor simulation well proved that PEG-His@BPC could first show good tumor penetration, and then retain in deep tumor for a longer time, which was consistent with the previous reports 37,38 .

ROS generation
First, we synthesized PEG-Phe@BPC as control nanoparticles, in which the histidine of PEG-His@BPC was replaced by phenylalanine (Phe). The PEG-Phe@BPC could not remove the PEG shell under pH 6.5 because the Phe could not be protonated. DCFH-DA was chosen as the ROS fluorescent probe to determine intracellular ROS generation. The results showed that no ROS was detected in the PBS with or without laser irradiation group, indicating laser irradiation itself could not affect ROS generation. With the presence of MMP-2 (þE) and laser irradiation (þL), PEG-His@BPC þ E þ L group showed strongest ROS fluorescence, implying that the transformation from spherical nanoparticles to nanofibers could obviously improve the ability to generate ROS, which was due to enhanced intracellular retention capacity (Fig. 3A). Moreover, there was no detectable ROS in the Ce6 group, but a small amount of ROS was detected in PEG-His@BPC group and PEG-Phe@BPC group. This result implied that BBR also had the ability to produce ROS, which was consistent with previous reports 39 . In order to further prove the ability of BBR to produce ROS, we synthesized PEG-His@Arg-PLGVRKLVFF-Ce6 (PEG-His@APC) that was without BBR. As we expected, the results showed that PEG-His@BPC þ L group could generate more ROS than PEG-His@APC þ L group, and there was no ROS in PEG-His@APC group, indicating BBR could indeed generate ROS (Fig. 3B). Therefore, the generation of ROS could further magnify when Ce6 and BBR were used in the tumor treatment together, which could establish a good theoretical foundation for the combination therapy of PDT and chemotherapy.

Cytotoxicity analysis
In order to elucidate the relationship between incubation concentration of drugs and cytotoxicity and select the appropriate administration concentration, MTT assays were carried out (Fig. 4B). After laser irradiation, the toxicity increased obviously when the concentration of Ce6 was above 4 mg/mL, which was consistent with previous reports 40 . Moreover, the PEG-His@BPC þ E þ L showed obvious cytotoxicity with lowest halfmaximal inhibitory concentration value (IC 50 ) among all groups, indicating that shape transformable nanoparticles could enhance the cytotoxicity. In order to further investigate the antitumor effect at cellular level, the CalceineAM/PI double staining was performed (Fig. 4A). The results showed that all the cells in the PBS group survived regardless of laser irradiation, indicating that laser irradiation alone did not lead to cell death. However, a small number of dead cells were observed in PEG-His@BPC þ E group, which ascribed to the presence of BBR. After laser irradiation, due to the large amount of ROS generated by Ce6, the toxicity of each group had a corresponding increase. It could be found that the cytotoxicity of PEG-His@BPC þ E þ L group was the strongest, which induced almost 100% of cell death. These results indicated that the shape transformable nanoparticles with chemo-photodynamic therapy achieved the remarkable cytotoxicity, which was consistent with the previous results of ROS generation experiment. In addition, it had been reported that both BBR and Ce6 could promote apoptosis 41e45 . In order to further investigate the effect of PEG-His@BPC on apoptosis promoting, PI/Annexin VeFITC double staining experiment was carried out. The results showed that free BBR and Ce6þL could induce early apoptosis by 9.91 AE 0.52% and 26.70 AE 2.67% and late apoptosis by 21.16 AE 2.93% and 24.08 AE 2.02%, respectively. In addition, PEG-His@BPC þ E þ L reached the best degree of apoptosis among all formulations, even higher than that of free Ce6þL, resulting in 34.32 AE 3.13% of early apoptosis and 40.25 AE 3.52% of late apoptosis, respectively. Compared with PBS group, PEG-His@BPC þ E þ L group increased early apoptosis by 29.72% and late apoptosis by 33.94%, respectively ( Fig. 4C and D). These results jointly demonstrated that the shape transformable nanoparticles with chemo-photodynamic therapy could achieve the best effect of apoptosis. This was consistent with the above results of ROS experiment and MTT, indicating the strategy of shape transformable nanoparticles with combination therapy showed strong therapeutic effect.

In vivo targeting evaluation
In order to evaluate the tumor targeting capacity of the nanomedicines in 4T1 breast tumor models, unresponsive PEG-Phe@BBR-RPGFGNKLVFF-Ce6 (PEG-Phe@BRC), pH-responsive PEG-His@BRC, MMP-2-responsive PEG-Phe@BPC and dual-responsive PEG-His@BPC were constructed. The changes of fluorescence signals in vivo were observed at 4, 12, 24, 36 and 48 h after intravenous injection by in vivo fluorescence imaging system (PerkinElmer, Fig. 5A). Four kinds of nanoparticles were widely distributed throughout the body at 4 h, regardless of whether they could respond to tumor microenvironment or not. Importantly, PEG-His@BPC and PEG-Phe@BPC could accumulate at the tumor site in 48 h, while the fluorescence signal of PEG-His@BRC and PEG-Phe@BRC showed a trend of rapid clearance after 24 h, indicating that shape transformable nanoparticles could be more prone to accumulate at tumor site. The tumor fluorescence signal of PEG-His@BPC was slightly stronger than that of PEG-Phe@BPC at 24 h, confirming that charge reversible strategy could enable nanoparticles to accumulate at tumor site more easily. According to the ex vivo imaging of major organs and tumors at 48 h (Supporting Information Fig. S5A and S5B) and semi-quantitative results ( Fig.  S5B and S5C), the tumor fluorescence intensity of PEG-His@BPC was 1.2, 3.5, and 2.9 times higher than that of PEG-Phe@BPC, PEG-His@BRC and PEG-Phe@BRC, respectively. All the above results showed that the dual-responsive nanoparticles with transformable shape and reversible charge could effectively enhance the tumor targeting in vivo, which was very important for the follow-up treatment effect and consistent with the previous reports 46 .

In vivo retention evaluation
In order to further confirm the tumor retention of shapetransformed nanoparticles in vivo, PEG-His@BPC, PEG-Phe@BPC, PEG-His@BRC and PEG-Phe@BRC were intratumorally injected into mice with the same tumor size. Then, the intratumoral fluorescence signals were captured at 0, 8, 12, 24 and 36 h, respectively ( Fig. 6A and B). The results showed that the intratumoral fluorescence signals of all groups were strong and similar at the initial time point. However, at 24 h, the intratumoral fluorescence signals of the PEG-His@BRC and PEG-Phe@BRC almost disappeared, while the intratumoral fluorescence signals of the PEG-His@BPC and PEG-Phe@BPC still maintained a very high level, and the intratumoral intensity of PEG-His@BPC was 1.79, 1.99 and 1.36 times higher than that of PEG-His@BRC, PEG-Phe@BRC and PEG-Phe@BPC, respectively. These results confirmed that transformable shape could enable nanoparticles more prone to retain at tumor site. The in vivo imaging results were consistent with the ex vivo . Data are presented as mean AE SD (n Z 3). *P < 0.05 and ***P < 0.001 are considered as significant difference. results (Fig. 6C). In order to further confirm that the enhanced tumor retention was due to the shape transformation of PEG-His@BPC, TEM (JEOL) was utilized to analyze the nanomaterials at the tumor site. Result showed that the PEG-His@BRC maintained a spherical structure in the tumor, while many nanofibers were found in the tumor treated with PEG-His@BPC (Supporting Information Fig. S6A and S6B), indicating that the enhanced tumor retention of PEG-His@BPC was due to the transformation to nanofibers.

Anti-tumor effect in vivo
Inspired by in vitro toxicity test and in vivo tumor targeting and tumor retention evaluation, the in vivo anti-tumor effect was evaluated. The results showed that the average tumor volume of PBS group, Ce6þL group, BBR group and PEG-His@BPC group increased rapidly, which was higher than 800 mm 3 on Day 22 (Fig. 7A-D). However, PEG-Phe@BPC þ L, PEG-His@BRC þ L, PEG-Phe@BRC þ L and PEG-His@APC þ L groups showed slower growth rates (Fig. 7E-I), and the PEG-His@BPC þ L group showed the smoothest growth curve. The average tumor volume of PEG-His@BPC group only reached 289 mm 3 (Fig. 7J). Compared with the PBS group on Day 22, the tumor inhibition rates of PEG-His@BPC, PEG-Phe@BPC, PEG-His@BRC, PEG-Phe@BRC and PEG-His@APC were 71.4%, 49.5%, 27.2%, 23.0% and 46.3%, respectively. The results indicated the dual-responsive nanoparticles with transformable shape and reversible charge could obviously enhance the therapeutic effect against tumor. The body weight of mice in each group did not change much (Fig. 7K). The results of tumor image and tumor weight under natural light were in line with the previous measurements ( Fig. 7L and Supporting Information Fig. S7). The images of hematoxylin and eosin (H&E) staining sections showed that there were no obvious histological changes in the main organs (Supporting Information Fig. S8). TUNEL staining and Ki67 staining showed that PEG-His@BPC had the best capacity to promote tumor apoptosis (Supporting Information Fig. S9A and S9B). The blood biochemical data also showed that the liver and kidney tissue function of each group was normal and there was no obvious toxicity ( Fig. S9C-S9G). These results jointly demonstrated that PEG-His@BPC not only amplified chemo-photodynamic therapy of breast cancer through the combination of shape transformation and charge reversal, but also had low toxicity to normal organs.

Conclusions
Herein, we reported a new type of dual-responsive nanoparticles (PEG-His@BPC) with shape transformation, reversible charge and chemo-photodynamic therapy. First, the negatively charged spherical PEG-His@BPC had long circulation time in the blood. When the PEG-His@BPC reached the tumor site through the EPR effect, the PEG shell fell off triggered by the acidic tumor microenvironment, resulting in charge reversal and exposure of the enzyme cutting site. The positive charge of the BPC was conducive to the tumor penetration. When the BPC penetrated into the deep tumor, BBR was released to promote apoptosis and the spherical BPC transformed into nanofibers, which could enhance the tumor retention. The shape transformation and its effect were confirmed by in vitro and in vivo analysis. In addition, PEG-His@BPC successfully amplified chemo-photodynamic therapy of breast cancer through the combination of shape transformation and charge reversal. In general, these studies have proved that this nanosystem was very promising in therapy of breast cancer.
The future research direction of dual-responsive nanoparticles with transformable shape and reversible charge should try to treat breast cancer brain metastases (BCBMs), which are cause of morbidity and mortality among patients with breast cancer 47,48 . Systemic drug therapy is usually effective for peripheral breast cancer, but it is often ineffective for BCBM because of poor penetration to the bloodebrain barrier (BBB) 49e51 . Some studies have shown that the shape of nanoparticles has obvious effect on the penetration to BBB, which suggested that altering the shape of nanocarriers could influence their uptake into the brain, whereby increasing the efficacy and bioavailability of therapeutic agents 52e54 . Moreover, a variety of strategies or nanoparticles have been found to improve the penetration to BBB 55 , such as the strategy of escaping from abluminal LRP1-mediated clearance 56 , PSMAtargeted nanoparticles 57 , LRP1-upregulated nanoparticles 58 . Combining shape transformable nanoparticles with above strategies or nanoparticles to overcome the BBB may be a new direction for defeating BCBM.