Co-administration of a charge-conversional dendrimer enhances antitumor efficacy of conventional chemotherapy

Accepted Manuscript Co-administration of a charge-conversional dendrimer enhances antitumor efficacy of conventional chemotherapy Jun Cao, Chenhong Wang, Leijia Guo, Zhiyong Xiao, Keliang Liu, Husheng Yan PII: DOI: Reference:

S0939-6411(17)31405-4 https://doi.org/10.1016/j.ejpb.2018.02.035 EJPB 12715

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Revised Date: Accepted Date:

5 December 2017 23 February 2018 25 February 2018

Please cite this article as: J. Cao, C. Wang, L. Guo, Z. Xiao, K. Liu, H. Yan, Co-administration of a chargeconversional dendrimer enhances antitumor efficacy of conventional chemotherapy, European Journal of Pharmaceutics and Biopharmaceutics (2018), doi: https://doi.org/10.1016/j.ejpb.2018.02.035

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Co-administration of a charge-conversional dendrimer enhances antitumor efficacy of conventional chemotherapy Jun Caoa, Chenhong Wang b, Leijia Guoa, Zhiyong Xiaob, Keliang Liub, Husheng Yana,c,* a

Key Laboratory of Functional Polymer Materials (Ministry of Education), College

of Chemistry, Nankai University, Tianjin 300071, China b

Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China

c

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),

Tianjin 300071, China * Corresponding author. E-mail: [email protected] Abstract:

Despite

extensive

investigations,

the

clinical

translation

of

nanocarrier-based drug delivery systems (NDDS) for cancer therapy is hindered by inefficient delivery and poor tumor penetration. Conventional chemotherapy by administration of free small molecule anticancer drugs remains the standard of care for many cancers. Herein, other than for carrying and releasing drugs, small nanoparticles were used as a potentiator of conventional chemotherapy by co-administration with free chemotherapeutic agents. This strategy avoided the problems associated with drug loading and controlled release encountered in NDDS, and was also much simpler than NDDS. Negatively charged poly(amido amine)-2,3-dimethylmaleic monoamide (PAMAM-DMA) dendrimers were prepared, which possessed low toxicity and can be converted to positively charged PAMAM dendrimers responsive to tumor acidic pH. The in situ formed PAMAM in tumor

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tissue promoted cellular uptake of co-administered doxorubicin by increasing the cell membrane permeability, and subsequently enhanced the cytotoxicity of doxorubicin. The small size of the dendrimers was favorable for deep penetration in tumor. Co-injection of PAMAM-DMA with doxorubicin into nude mice bearing human tumors almost completely inhibited tumor growth, with a mean tumor weight reducing by 55.9% after the treatment compared with the treatment with doxorubicin alone. Keywords: Chemotherapy potentiator; Charge conversion; Dendrimer; Drug delivery; pH responsive

1. Introduction Cancer is one of the leading causes of morbidity and mortality worldwide [1]. Conventional chemotherapy combined with radiotherapy and surgical resection has contributed greatly to reducing cancer mortality rates. However, the severe toxic side effects of and multi-drug resistance (MDR) to free chemotherapeutic agents are major impediment for successful chemotherapy [2]. Especially, tumors tend to become resistant to drugs during the course of therapy, consequently drug resistance contributes to therapeutic failure and tumor recurrence in over 90% of patients, becoming the major obstacle in the chemotherapeutic treatments of many human cancers [3,4]. A major mechanism of MDR has been regarded as the over-expression of

ATP-binding

cassette (ABC)

transporters,

such as

plasma

membrane

P-glycoprotein, which are able to pump various drugs out of the cells, causing a too

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low intracellular concentration of drug to kill the cells [5,6]. To counter the drug efflux from cancer cells, higher doses of chemotherapeutic agents are theoretically required. Unfortunately, the therapeutic window of anticancer agents is very narrow, as even a slight increase in chemotherapy dosages results in potentially lethal side effects [6]. To overcome the systemic toxicity and adverse effects, and to circumvent the MDR of conventional chemotherapy, nanocarrier-based drug delivery systems have been extensively explored during the past few decades [7-10]. Systemically administered nanoparticles have longer circulation times and can passively accumulate in the tumors by the enhanced permeability and retention (EPR) effect, and subsequently drug is delivered in tumor. It is expected that nanocarrier-based drug delivery systems can enhance the safety and efficacy of existing anticancer therapeutics, thereby improving therapeutic outcomes and minimizing undesirable side effects, as compared to the free drug. Despite massive investments and a large number of scientific publications regarding nanocarrier-based drug delivery, only a handful of clinical products exist today, and these platforms show only modest efficacious patient responses and marginal improvements over conventional formulations [11-14]. This status can be attributed to two major obstacles in the development of nanocarrier-based drug delivery systems: low drug delivery efficiency and poor tumor penetration of nanoparticles [12,13]. The drug delivered at tumor sites or within tumor cells often cannot reach the therapeutic concentration level to kill cancer cells due to low delivery efficiency. And even worse, a prolonged exposure of

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cancer cells to a therapeutic agent at a low concentration level can induce acquired drug resistance [15]. The low delivered drug amount to target cells by nanocarrier-based delivery systems can be resulted from one or more of the following factors: a low drug loading capacity (in particular for nanocarriers with small sizes), short blood circulation time, premature release, inadequate cellular uptake, and non-immediate and incomplete release in the target cells [7,12,13]. Even if a small fraction of nanoparticles with a size around the optimal size for EPR effect and tumor accumulation (~100 nm [16]) accumulates in solid tumors through the EPR effect, they are mainly restricted to the adjacent regions of tumor vasculatures after extravasation due to the dense and heterogeneous extracellular matrix and high interstitial fluid pressure, leading to reduced efficacy and the development of drug resistance owing to the exposure of the cancer cells in deep sites to a low drug concentration level [17,18]. Herein we attempted an alternative strategy using nanoparticles to enhance the efficacy of conventional chemotherapy. Other than for carrying and releasing drugs as in nanocarrier-based drug delivery systems, nanoparticles were used as an enhancer to promote the internalization of free drugs into cancer cells during the conventional chemotherapeutic treatment, thereby increasing the drug concentration in and countering the drug efflux caused by ABC transporters from cancer cells, and subsequently improving the efficacy of the conventional chemotherapy. In this strategy, the problems associated with drug loading and controlled release in nanocarrier-based drug delivery systems are avoided. This strategy is also much

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simpler than nanocarrier-based drug delivery systems. The simplicity of this system would be favorable for clinical translation. 2. Materials and methods 2.1. Materials Methanol, ethylenediamine (Tianjin Chemical Company, China) and methyl acrylate (J&K Scientific Ltd) were distilled before use. 2,3-Dimethylmaleic anhydride (DMA) was purchased from Heowns Biochemical Technology Co., Ltd (Tianjin, China) and used without further purification. Doxorubicin hydrochloride (DOX· HCl) was purchased from Beijing HVSF United Chemical Materials Co., Ltd (Beijing , China). The human breast cancer cell line, MCF-7, was purchased from the Cell Resource Center of Peking Union Medical College (Beijing, China). Female BALB/c nude mice (4–6 w, 18–20 g) were purchased from SPF (Beijing ) Biotechnology Co., Ltd (Beijing, China). 2.2. Synthesis of PAMAM dendrimers PAMAM dendrimers with ethylenediamine as the initiator core were synthesized by alternating exhaustive Michael addition of methyl acrylate and an exhaustive amidation of the resulting ester with ethylenediamine according to our previously reported procedure [19]. Briefly, a solution of ethylenediamine (0.41 g, 6.75 mmol) in methanol (15 mL) was stirred at 0C under a nitrogen atmosphere for 30 min. Methyl acrylate (11.94 g, 138.7 mmol) was added slowly and the mixture was bubbled with nitrogen for 30 min under cooling, and then was stirred for 12 h at room temperature. The resulting mixture was evaporated at 50C under reduced pressure to remove the

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solvent and unreacted methyl acrylate to get generation 0.5 (G0.5) PAMAM. G0.5 PAMAM (2.727 g, 6.75 mmol) was dissolved in methanol (10 mL) and the solution was stirred at 0oC under a nitrogen atmosphere for 30 min. Ethylenediamine (9 g, 150 mmol) was added and the mixture was bubbled with nitrogen for 30 min under cooling and was stirred 24 h at 25C. The resulting mixture was evaporated at 50C under reduced pressure to remove the solvent and unreacted ethylenediamine to get generation 0 (G0) PAMAM. G1G7 PAMAM dendrimers were similarly synthesized by an exhaustive Michael addition of methyl acrylate followed by an exhaustive amidation of the resulting ester with ethylenediamine from the previous generation PAMAM. The G2G7 PAMAM dendrimers were purified by dialysis against methanol for 48 h by using dialysis membranes with molecular weight cut-off values of 3,000 Da, 5,000 Da and 12,000 Da, respectively. 1H NMR spectrum of G4 PAMAM as an example is shown in Fig. S1A. 2.3. Synthesis of DMA-modified PAMAM dendrimers DMA-modified PAMAM (PAMAM-DMA) was synthesized by the reaction of PAMAM with 2,3-dimethylmaleic anhydride (DMA) following to a literature method with modifications [20]. Typically, a solution of G4 PAMAM (0.21 g, 0.96 mmol terminal amine groups) in distilled water (10 mL) was stirred at 0C for 30 min. To this solution, DMA (0.18 g, 1.44 mmol) was added in five portions under stirring in an ice/water bath, during which the pH of the solution was maintained to be ~ 8.5 by adding aqueous NaOH (1 M). The mixture was further stirred for 30 min under

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cooling and then at room temperature overnight. The resulting solution was transferred to a dialysis tube with molecular weight cut-off of 12,000 Da and dialyzed against distilled water for 48 h. The product, G4 PAMAM-DMA, was obtained after lyophilization. 1H NMR spectrum of G4 PAMAM-DMA is shown in Fig. S1B. Compared to G4 PAMAM, the peaks at 1.76 and 1.81 ppm appeared and were assigned to the methyl groups of the resulting amides. From the integration ratio of the peaks at 2.38 ppm and 1.701.85 ppm, it was estimated that approximately 95% terminal amine groups of the PAMAM were converted to amides. G5 PAMAM-DMA, G6 PAMAM-DMA and G7 PAMAM-DMA were synthesized similarly with the same molar ratio (1/1.5) of terminal amine groups of the dendrimer to DMA. 2.4. Analyses of particle sizes and  potentials Particle sizes and  potentials were measured by dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern, UK) with a fixed scattering angle of 90 at 25C. 2.5. Cell Culture MCF-7 cells were grown and maintained in Roswell Park Memorial Institute 1640 (RPMI-1640; Gibco, USA) growth medium containing 10% fetal bovine serum (FBS; Hyclone, USA) and 1% penicillin/streptomycin (Invitrogen, USA) at 37 oC in a humidified atmosphere with 5% CO2. 2.6. Cellular uptake assay MCF-7 cells were seeded in 12-wells cell culture plates at a density of 1  105 cells per well in 1 mL of complete medium and were incubated at 37C for 24 h. After

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removing the culture medium, DOX (dissolved in serum-free medium) or PAMAM plus DOX (dissolved separately in serum-free medium) was added into the plates. The final concentrations of DOX and PAMAM were 4 g/mL and 25 g/mL, respectively. After incubation at 37C for 0.5 h, the medium was removed. The cells were washed with PBS twice and harvested with 0.25% (w/v) trypsin/0.02% (w/v) EDTA solution, then were transferred to fluorescence-activated cell sorting (FACS) tubes (Becton Dickinson). Cells were analyzed with Facs-Calibur (Becton Dickinson, USA) at an excitation wavelength of 488 nm (FL2 channel). A total of 10,000 cells of sample were analyzed. The number of cells with fluorescence intensity higher than that of unlabeled cells was used to determine the fraction of labeled cells. The baseline was obtained by analyzing unlabeled control cells. MCF-7 cells were treated with DOX or PAMAM-DMA plus DOX and cellular uptake was assayed in the same way as shown above except that the final concentration of PAMAM-DMA was 50 g/mL and the incubation time was 2 h. 2.7. Cytotoxicity assay MCF-7 cells were seeded in 96-wells plates at a density of 5  103 cells per well and were incubated at 37C for 24 h. The cells were washed twice with PBS. After removing the culture medium, DOX (dissolved in serum-free medium), PAMAM (dissolved in serum-free medium) or PAMAM plus DOX (dissolved separately in serum-free medium) was added into the plates. After incubation at 37C for 0.5 h, the medium was replaced with fresh medium and the cells were incubated for another 24 h. The cells were washed three times with PBS and then the medium was replaced by

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80 μL of PBS and 20 μL of Cell-Titer 96®AQueous One Solution Reagent. After 3 h at 37C, optical density (OD) values were measured at 490 nm using a SpectraMAX® MS microplate reader (Molecular Devices, Sunnyvale, CA, USA). Serum-free medium was used as a negative control. The cell viability of the sample was calculated by ODsamlple/ODcontrol  100%. This experiment was repeated five times for each sample. The cytotoxicity of DOX, PAMAM-DMA and PAMAM-DMA plus DOX against MCF-7 cells was assayed similarly as shown above except that the final concentration of PAMAM-DMA was 50 g/mL,

incubation time was 2 h, and pH of the medium

in which drug and/or dendrimer were dissolved for assay was 7.4 or 6.5. 2.8. On-time Scanning Confocal Microscope Analysis MCF-7 cells were seeded in a Lab-Tek® chamber slide system with 8 wells (Thermo Fisher Scientific Inc.) with a cell density of 1 × 10 4 cells per well. After incubation for 24 h, the medium was removed and the cells were washed with PBS twice. Cells were stained in 0.2 µM calcein (TCI, Tokyo, Japan). After the medium was discarded, the cells were washed with PBS for three times. To the plates, 100 µL of DOX (4 g/mL) or DOX (4 g/mL) plus PAMAM (25 g/mL) in PBS with the preset pH value was added for imaging. The imaging process was recorded on a Perkin Elmer Confocal System Ultra VIEW VOX with excitation at 488 nm (calcein) and 561 nm (DOX). 2.9. In vivo tumor growth inhibition MCF-7 models were used to evaluate the therapeutic efficacy of formulations. In

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the breast xenograft tumor model, 3 × 106 MCF-7 cells in 200 μL PBS were inoculated subcutaneously (s.c.) at the right flank of female BALB/c nude mice (4–6 w, 18–20 g). The volume of tumor (V) was measured based on the equation: V = a × b2/2, where a and b are the length and width of the tumor, respectively. When the tumor reached approximately 200 mm3, the tumor-bearing mice were randomly divided into four groups (n = 5). At days 0 and 7, DOX (5 mg/kg), DOX (5 mg/kg) plus G5 PAMAM-DMA (15 mg/kg), G5 PAMAM-DMA (15 mg/kg) in PBS, or saline (control) was administered by intraperitoneal injection in the mice. The tumor size and body weight were monitored daily after the first injection. All the treated mice were sacrificed at day 14. The collected tumor tissues were weighed and analyzed by hematoxylin–eosin (H&E) and TUNEL assays. The animal test was approved by the Animal Care and Use Committee of the Beijing Institute of Pharmacology and Toxicology. 2.10. Statistical analysis Statistical analysis of variance (ANOVA) with the Tukey post-hoc test and two-tailed unpaired Students’ t-test were utilized to evaluate the differences between treatment groups with p < 0.05 as statistically significant. The results were shown as mean ± standard deviation. 3. Results and discussion 3.1. Enhanced cellular uptake of doxorubicin by dendrimers It has previously been reported that positively charged nanoparticles including dendrimers disturb or disrupt negatively charged biological membranes, resulting in

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an increase in membrane permeability [21-23]. This prompted us to study the possibility of using positively charged nanoparticles as the promoter of drug internalization due to the enhanced permeability. To prove such assumption, we used positively charged poly(amido amine) (PAMAM) dendrimers as the promoter. Human breast cancer cells (MCF-7) with entrapped calcein were incubated with DOX alone, or with DOX plus generation 4 (G4) PAMAM. Confocal laser scanning microscopy (CLSM) images of the cells after incubation are shown in Fig. S2. Indeed, the dendrimer lead to a quick leakage of the entrapped calcein out of cells and an increase in DOX concentration within cells after the incubation. These results indicated that the permeability of cell membranes increased after treatment with G4 PAMAM. The mechanism of the cell membrane disruption by positively charged particles has been extensively investigated and is attributed to the interaction between the positively charged particles and the negatively charged phospholipid bilayers of cell membrane [21-23]. The enhanced cellular uptake of DOX into MCF-7 cells upon co-incubation with generations 4 to 7 (G4G7) PAMAM dendrimers was further quantitatively investigated by flow cytometry and the results are shown in Fig. 1A. It was observed that PAMAM dendrimers of all generations tested enhanced DOX internalization into MCF-7 cells. The enhanced efficiency levels decreased roughly with increasing dendrimer generations, with 3.8-, 2.9-, 1.6- and 1.9-fold increases, respectively, after co-treatment with DOX plus G4, G5, G6 and G7 PAMAM dendrimers, compared with that after treatment with DOX alone. This decrease in the extents of the

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efficiency enhancement with the generations may be contributed to the decrease in molar concentrations at the same mass concentration used in the assay for all PAMAM dendrimers (the molecular weight of a PAMAM dendrimer is roughly double that of the previous generation of PAMAM). The cell viability of MCF-7 cells after incubation with DOX alone, with DOX plus PAMAM dendrimers, and with PAMAM dendrimers alone are presented in Fig. 1B. The group of the combination of DOX and PAMAMs showed the highest cytotoxicity for the dendrimers of all generations. Especially G4 PAMAM dendrimer markedly enhanced the cytotoxicity of DOX upon co-incubation. The effect of dendrimer generations on the extent of the enhanced cytotoxicity was roughly consistent with the effect of dendrimer generations on the cellular uptake (Fig. 1A). PAMAMs themselves also showed high cytotoxicity (Fig. 1B). This DOX cytotoxicity enhancement by PAMAM dendrimers after co-incubation should be resulted from both of the enhanced DOX internalization and cytotoxicity of PAMAM dendrimers themselves. It should be noted that the effect of the generations of PAMAM dendrimers on the cytotoxicity seems inconsistent with previously reported results [24,25]. This should be resulted from the factor that the same mass concentration of PAMAM dendrimers of different generations was used in cytotoxicity assay in this work, while the same molar concentration of different generations was used in the previous reports for comparison [24,25]. Although PAMAM dendrimers significantly enhanced the cytotoxicity of DOX after co-treatment, the dendrimers alone also had severe cytotoxicity as shown above,

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which would be harmful to normal cells as well. The toxicity of positively charged nanoparticles include dendrimers have also been reported previously [19,26-28]. To overcome the problem associated with the toxicity of the positively charged dendrimers to normal tissues while remaining their property capable of enhancing free drug internalization, PAMAM dendrimers were modified with 2,3-dimethylmaleic anhydride (DMA), forming more biocompatible negatively charged dendrimers (PAMAM-DMA). It has previously been reported that nanoparticles with neutral or negatively charged surfaces show low toxicity and prolonged blood circulation time [19,29-31]. For our system, we expected that, once localized to tumors, PAMAM-DMA would be converted to positively charged PAMAM dendrimers due to acidic pH-triggered hydrolysis of the -carboxylate amide (Scheme 1) because the extracellular pH value in most clinical tumors is more acidic (pH 6.5–7.0) than in healthy tissues (ca. pH 7.4) [32,33]. It has been shown that -carboxylate amide is stable at normal physiological pH but hydrolyzed at tumor acidic pH [20,34]. The charge conversion was confirmed by analysis of  potentials of PAMAM-DMA dendrimers after incubation in media at different pH values (Fig. 2). The  potentials of PAMAM-DMAs of all generations showed negative values in the early stage and gradually approached zero or a slightly positive value after incubation at pH 7.4, suggesting that the zwitterionic surfaces would gradually generate during blood circulation. It is previously reported that nanoparticles with zwitterionic surfaces exhibit prolonged blood circulation and superior biocompatibility [35,36]. Fig. 2 shows that, in contrast to the  potentials remaining negative or slightly positive upon

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incubation at pH 7.4, the  potentials of PAMAM-DMA dendrimers changed from negative to highly positive after incubation for a certain time at more acidic pHs. For example, the  potentials of G5 PAMAM-DMA were -10, +4 and +10 mV after incubation for 2 h at pH 7.4, 6.8 and 6.5, respectively. The hydrodynamic size distributions of PAMAM-DMA dendrimers and their hydrolyzed products after incubation at different pHs were determined by dynamic light scattering (DLS) and the results are shown in Fig. S3. The average hydrodynamic diameters of the dendrimers are shown in Table S1. The size of the dendrimers at pH 7.4 ranged from ~10  ~17 nm. Although the size of the dendrimers is smaller than the optimal size for EPR effect and tumor accumulation (~100 nm [16]), the small size should be favorable for deep penetration of the dendrimers into tumor tissues, and the deeper penetration would be furthermore favorable for the acidic pH-triggered charge conversion because the microenvironment in the inner tumor regions is more acidic than that in the perivascular space, where the pH is near neutral [32,37]. For the balance between renal clearance and tumor retention, it is shown that nanoparticles with a hydrodynamic diameter of ~10 nm is an excellent choice [38].

We used G5 PAMAM-DMA, which was ~11 nm in hydrodynamic

diameter, in in vivo assay as shown below. When the dendrimer became gradually positively charged responding to tumor acidic microenvironment, they may penetrate deeper in tumor tissues because it has been reported that cationic nanoparticles possess high potential for intratumoral penetration over neutral or anionic particles [39-43], and slightly positively charged nanoparticles penetrate deeper in tumor than

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highly positively charged ones [42,43]. The promotion of cellular uptake of DOX by PAMAM-DMA dendrimers was evaluated by CLSM (Fig. S4) and flow cytometry (Fig. 3A). It can be seen from Fig. 3A that PAMAM-DMAs had almost no effect on DOX internalization after co-incubation at pH 7.4. In contrast, PAMAM-DMAs significantly enhanced DOX internalization after co-incubation at pH 6.5. G5 PAMAM-DMA showed the highest extent of the enhanced internalization efficiency after incubation at pH 6.5, which was slightly different from the effect of the generation on the extent of the enhanced internalization efficiency by the corresponding PAMAMs, with G4 PAMAM showing the highest extent of the enhanced internalization efficiency (Fig. 1A). This may be a result of the lower positive charge densities of the dendrimers produced via partial cleavage of the -carboxylate amide after 2 h incubation (cf. Fig. 2) compared to corresponding PAMAMs. The highest extent of the enhanced internalization efficiency by G5 PAMAM-DMA should be a result of co-influence of the positive charge density and molar concentration. Fig. 3B shows the cell viability of MCF-7 cells after incubation for 2 h at pH 7.4 and 6.5 with DOX alone, with DOX plus PAMAM-DMAs, and with PAMAM-DMAs alone. Similar to the case of the incubation with PAMAMs (Fig. 1B), the combination of DOX and PAMAMs showed the highest toxicity for all PAMAM-DMAs at pH 6.5. G5 PAMAM-DMA exhibited the highest extent of cytotoxicity enhancement, which was in consistent with the results of the enhancement of DOX internalization (Fig. 3A). On the other hand, PAMAM-DMAs alone showed much lower cytotoxicity than

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PAMAMs with the same generation at pH 7.4. For example, the cell viability of MCF-7 cells after incubation with G5 PAMAM-DMA for a 4-fold longer time at pH 7.4 was still higher than that after incubation with G5 PAMAM (comparing Fig. 1B and Fig. 3B). 3.2. In vivo tumor growth inhibition assay The therapeutic efficacy of a combination of free DOX and G5 PAMAM-DMA was finally investigated via an in vivo tumor growth inhibition assay. Nude mice bearing subcutaneous MCF-7 tumors were treated by intraperitoneal (i.p.) injection with DOX alone, with DOX plus G5 PAMAM-DMA, and with G5 PAMAM-DMA alone at days 0 and 7, and the results are shown in Fig. 4. The tumor from the saline group grew rapidly, with a mean tumor volume increasing by 13.6 times at day14. Administration of DOX alone markedly inhibited the tumor growth, with the mean tumor volume decreasing by 57.7% and the mean tumor weight decreasing by 55.4% at day 14 in comparison with the saline group. However, the mean tumor volume still increased by 5.5 times at day 14 compared to that at day 0. Co-administration of DOX with G5 PAMAM-DMA further markedly inhibited the tumor growth, almost completely inhibiting tumor growth (Figs. 4A, 4B and 4C). The co-injection of the dendrimer caused a 76.9% decrease in the mean tumor volume (Fig. 4A) and a 55.9% decrease in the mean tumor weight (Fig. 4C) compared to the DOX alone group at day 14. These results indicated that co-injection of the dendrimer significantly enhanced the anticancer efficacy of DOX. G5 PAMAM-DMA alone also showed slight tumor growth inhibition.

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Fig. 4D shows the body weight changes after treatment. The mean body weight of mice treated with saline slightly decreased at day 14 compared with that at day 0. In comparison, the mean body weight significantly decreased after treatment with DOX alone. These results indicated that both of the tumor growth and the toxicity of DOX to healthy tissues should lead to the body weight loss, and the DOX treatment had more marked effect on the body weight reduction than the tumor growth, suggesting the severe side effect of DOX. The mean body weight loss of mice co-treated with DOX and G5 PAMAM-DMA was lower than that of mice treated with DOX alone.

This observation can be explained by the fact that the less tumor

growth in the co-treatment group makes the mean body weight decrease less compared with the DOX alone group. Fig. 4D also shows that the body weight of the co-treatment group began to increase 4 days later after the second injection (at day 7). In contrast, the body weight of the DOX alone group began to increase 6 days later after the second injection. These results indicated that the health of the mice treated with DOX plus G5 PAMAM-DMA recovered better than that of the mice treated with DOX alone in the judgment by the body weight changes. The treatment with G5 PAMAM-DMA alone caused almost the same mean body weight change as the saline group, indicating a low toxicity of the dendrimer to healthy tissues. 3.3. Tumor histology analysis The cell proliferation and apoptosis in the tumor tissues after treatment were evaluated by hematoxylin and eosin (H&E) and terminal deoxynucleoitidyl transferase (TUNEL) assays as shown in Fig. 5. The H&E histologic images showed

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the highest massive cancer cell remission and the voidest spaces in the tumor tissue after treatment with DOX plus G5 PAMAM-DMA compared to the other groups. The TUNEL results also showed that the DOX plus G5 PAMAM-DMA group possessed the highest level of cell apoptosis. 4. Conclusions In summary, while positively charged PAMAM dendrimers can promote internalization of DOX into cancer cells, they also showed toxicity to normal tissues. Therefore, negatively charged PAMAM-DMA dendrimers were prepared and they showed low cytotoxicity in normal/neutral environment. Once accumulated in tumor tissues, PAMAM-DMA dendrimers can be converted to positively charged PAMAM dendrimers responsive to tumor acidic microenvironment, subsequently promoting DOX internalization. Co-administration of DOX plus G5 PAMAM-DMA in mice bearing MCF-7 tumors significantly enhanced the efficacy of tumor growth inhibition compared with administration of DOX alone. The system reported in this work is simpler, avoiding the problems of drug loading and controlled release encountered in nanocarrier-based delivery systems. The advantages of the simplicity and high potentiation level provide this system with promising potentials for clinical applications Acknowledgements This work was supported by National Natural Science Foundation of China (51373080) and PCSIRT (IRT1257) Appendix A. Supplementary data

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Scheme 1. Schematic illustration of charge conversation.

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Fig. 1. Flow cytometry analysis (A) and cell viability (B) of MCF-7 cells incubated with DOX (4 g/mL) alone, a combination of DOX (4 g/mL) and PAMAM dendrimers (25 g/mL), and PAMAM dendrimers (25 g/mL) alone for 30 min.

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Fig. 2.  potentials of dendrimers after incubation at various pHs for different times.

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Fig. 3. Flow cytometry analysis (A) and cell viability (B) of MCF-7 cells incubated with DOX (4 g/mL) alone, a combination of DOX (4 g/mL) and PAMAM-DMA dendrimers (50 g/mL), and PAMAM-DMA dendrimers (50 g/mL) alone for 2 h.

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Fig. 4. Enhanced antitumor efficacy of DOX with co-injection of G5 PAMAM-DMA. Mice bearing MCF-7 tumors were injected with DOX alone (5 mg/kg), DOX (5 mg/kg) + G5 PAMAM-DMA (15 mg/kg), and G5 PAMAM-DMA (15 mg/kg) alone at day 0 and day 7. Changes in tumor volumes (A) and body weights (D) of the mice were measured daily. The tumors isolated at day 14 were photographed (B) and weighed (C). Data are shown as the mean ± SD (n = 5). * p < 0.05, ** p < 0.001, *** p < 0.0001.

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Fig. 5. Histological images of tumor sections after the administrations of saline, G5 PAMAM-DMA, DOX, and DOX plus G5 PAMAM-DMA. Cells were stained with H&E and TUNEL (blue, DAPI-stained nuclei; green, TUNEL-stained apoptotic cells).

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