Polymer–Drug Nanoparticles Combine Doxorubicin Carrier and

Dec 27, 2016 - Key Laboratory of Drug Targeting and Drug Delivery Systems, West China ... and metastasis areas of lungs 18 days after treatment (PDF)...
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Polymer-drug nanoparticles combine doxorubicin carrier and heparin bioactivity functionalities for primary and metastatic cancer treatment Ling Mei, Yayuan Liu, Chunyu Xia, Yubei Zhou, Zhirong Zhang, and Qin He Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.6b00979 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016

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Molecular Pharmaceutics

Polymer-drug nanoparticles combine doxorubicin carrier and heparin bioactivity functionalities for primary and metastatic cancer treatment Ling Mei, Yayuan Liu, HuaJin Zhang, Zhirong Zhang, Huile Gao and Qin He* Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University. No. 17, Block 3, Southern Renmin Road, Chengdu 610041, China *Corresponding author E-mail: [email protected] Tel/Fax: +86-28-85502532. Abstract Here, a biocompatible amphiphilic copolymer of low molecular weight heparin (LMWH) and doxorubicin (DOX) connected by an acid-sensitive hydrazone bond for enhanced tumor treatment efficacy and safety has been designed and tested. The conjugate combines DOX delivery with LMWH anti-metastatic capabilities. After the nanoparticles reach the tumor site, the acidic tumor micro-environment triggeres the breakage of the hydrazone bond releasing DOX from the nanoparticles, which resulted in an increase in the cellular uptake and enhanced in vivo antitumor efficacy. A 3.4-fold and 1.5 fold increase in tumor growth inhibition were observed compared to the saline-treated control group and free DOX-treated group respectively. The LMWH-based nanoparticles effectively inhibited interactions between tumor cells and platelets mediated by P-selectin reducing metastasis of cells both in in vitro and in vivo models. The improved safety and therapeutic effect of LMWW-DOX nanoparticles offers new potential for tumor therapy. KEYWORDS: drug delivery; metastasis; self-assembly; low molecular weight heparin; pH sensitive 1. Introduction Nanoparticles have received increasing attention in drug delivery due to their lower systemic toxicity and enhanced tumor distribution1. Hence, numerous multifunctional nanoparticles have emerged in recent decades2, 3 that have been designed with properties that allow them to escape the immune system, prevent the degradation of bioactive molecules, target specific subcellular organelles, or trigger drug release in response to the microenvironment4-6. Some of the improvements are considered necessary for drug delivery; however, excessive modifications often form obstacles during clinical application7, and the toxicity of the materials has become an 1

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increasing concern. Most studies that focus on nano-based drug carriers ignore the cytotoxicity of drug carriers themselves8. Because carrier materials always display poor metabolism and elimination properties, it is important to construct drug delivery systems with both efficient targeting ability and a safer application. In fact, in the mid-1970s, Ringsdorf et al. proposed the concept of covalently attaching chemotherapeutic agents to a water-soluble polymer9. This type of system was thought to modulate the pharmacokinetics of the drug attached to the polymeric carrier and release more drug molecules at the disease site. Soon afterward, this type of self-assembled conjugate became a rapidly growing field10, 11. Similarly, to solve the problem of potential toxicity of carriers caused by poor metabolism and elimination, Ying’s group developed nanocomplexes consisting of protein drugs and green tea catechin derivatives, demonstrating greater safety and better antitumor effects12. The common idea behind these studies is that the drug delivery systems all comprised of only active components, and the absence of carrier materials should lead to a safer clinical application. Superior to artificial materials, certain types of natural polysaccharides, such as hyaluronic acid13, chitosan14 and heparin15, 16 have inspired the formulation of nanoparticles due to their nontoxic and nonimmunogenic properties17. Unfractionated heparin (UFH) is one of the most unique materials. In our previous study18, it exhibited inhibitory effects on tumor angiogenesis and metastasis in addition to anticoagulant activity, which made it different from other polysaccharides. It has been a long time since the clinical application of UFH as an anticoagulant in 1937. However, some side effects, such as thrombocytopenia and hemorrhagic complications, have limited its clinical use19. To remedy this issue, low molecular weight heparin (LMWH), which was obtained from the depolymerization of UFH, has been applied as a good alternative for UFH20. Although the development of improved tumor therapies benefited from nanotechnology, few effective strategies could inhibit tumor metastasis. Together, ideal treatment must integrate an inhibitory effect on metastatic tumor cells and cytotoxicity in one drug delivery system (DDS). To exploit effective DDSs, which are expected to prevent tumor growth, metastasis and potentially translate into the clinic, we report tumor acidic environment-responsive amphiphilic nanoparticles based on LMWH and DOX (Fig. 1). We hypothesize that low molecular weight heparindoxorubicin (LH-DOX) has carrier, anti-cancer and anti-metastatic functionality. The drug DOX is gathered in the hydrophobic core and is protected by hydrophilic LMWH, which could reduce opsonization and lengthen the circulation time in the blood. After accumulation in tumor tissues, DOX could be released in an acidic micro-environment and kill tumor cells effectively21. LH-DOX is different from previous systems. First, compared with UFH, LMWH has been reported to possess comparable or even better anticoagulation, anti-angiogenesis and anti-metastatic efficacy22. Due to the lower molecular weight and smaller molecular weight distribution, we removed the hydrophobic group of deoxycholic acid because LH-DOX could simply self-assemble into a nanoparticle. In 2

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Molecular Pharmaceutics

addition, the relatively narrow molecular weight distribution of LMWH compared to UFH might cause the size and polymer dispersion index (PDI) of nanoparticles to decrease, which means the size distribution of the nanoparticle was more uniform. Second, the toxicity was evaluated using various assays related to safety profiles. The cytotoxicity was investigated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assays, and the interaction between nanoparticles and erythrocytes was evaluated by a hemolytic test. The long-term toxicity to mice was tested with hematological analysis and serum biochemistry tests. UFH caused definite heparin-induced thrombocytopenia with a decreased platelet count, while LMWH did not. Hence, LMWH and LH-DOX had no effect on the blood cell counts of mice. Above all, as a widely implemented anticoagulant drug, LMWH exhibited the same potent anti-metastatic efficacy as UFH. In addition, we further explored the related mechanisms. Previous studies have verified that platelets could promote tumor cells to gain migration and invasion ability18. In this study, we demonstrated that LMWH could inhibit the adhesion between tumor cells and platelets, which was mediated by P-selectin.

Fig.1. Schematic design of the multifunctional polymer-drug conjugate.

2. Experimental 2.1. Materials Low-molecular weight heparin (Enoxaparin sodium, LMWH) was purchased from Melonepharma (Dalian, China). 6-Aminocaproic acid, thionyl chloride, 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) were purchased from Keddia Reagent (Chengdu, 3

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China). Doxorubicin hydrochloride was obtained from Beijing Huafeng United Technology Co., Ltd. (Beijing, China). BD Matrigel was purchased from BD Biosciences (San Jose, CA, USA). MTT, 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DiD), 4, 6- diamidino-2-pheylindole (DAPI) and calcein-AM were purchased from Beyotime Institute Biotechnology (Shanghai, China). Carboxyfluorescein succinimidyl amino ester (CFSE) was purchased from Dojindo (Kumamoto, Japan). Rabbit anti-CD41 antibody and rabbit anti-CD62p antibody were purchased from Zen Bioscience (Chengdu, China). Plastic cell culture dishes and plates were purchased from Wuxi NEST Biotechnology Co. (Wuxi, China). Other chemicals were obtained from Sinopharm Chemical Reagent (Shanghai, China) and were of analytical grade. Murine melanoma cells (B16F10) and breast cancer cells (4T1) were kindly obtained from State Key Laboratory of Biotherapy (Sichuan University) and cultured in DMEM and RPMI-1640 medium (GIBCO), respectively, at 37 °C. BALB/c mice and C57/BL6 mice (20 ± 2 g) were purchased from Dashuo Biotechnology Co., Ltd, (Chengdu, China), and all animal experiments were performed in accordance with the rules of the ethics committee of Sichuan University. 2.2. Synthesis of LH-DOX In brief, to synthesize cleavable LH-DOX, ethyl 6-aminohexanoate hydrochloride was first prepared, as described previously18, by coupling 6-aminocaproic acid and ethanol. Then, the ester bond was reacted with hydrazine to form hydrazide. The product was further reacted with DOX to form a hydrazone linker and analyzed by 1HNMR. Finally, the product was bound to different amounts of LMWH via amide linkage in the presence of EDC and NHS to form cleavable LH-DOX. The reaction solution was dialyzed exhaustively with ultrapure water using a dialysis membrane (molecular weight cut-off (MwCO) = 1000) in the dark. The retentate was lyophilized to give a red solid. Meanwhile, non-cleavable LH-DOX was obtained by coupling the carboxyl groups of LMWH and the amino groups of DOX. The reaction was performed in the dark for 24 h, followed by dialysis and lyophilization. All the structures were characterized by 1HNMR spectra using a Varian Mercury 400 NMR system (Varian Inc., Palo Alto, CA, USA). 2.3. Preparation and characterization of LH-DOX The self-assembled nanoparticles were obtained by dissolving the conjugate LH-DOX in phosphate buffer saline (PBS) buffer (pH=7.4) with gentle shaking. The solution was then sonicated using a probe-type sonifier (JY92-IIN, Ningbo Scientz Biotechnology CO,.LTD, Ningbo, China) at 200 W with 15 s pulses. Particle size and zeta potential were determined by dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano ZS (Malvern, UK). The morphology of the nanoparticle was captured by transmission electron microscopy (TEM) (JEM-100CX, JEOL, Japan). The content of DOX in LH-DOX was detected by a Varioskan Flash Multimode Reader (Thermo, USA) at Ex = 470 nm and Em = 590 nm. The critical micelle concentration (CMC) of LH-DOX was determined in water 4

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Molecular Pharmaceutics

using pyrene as a fluorescent probe. The fluorescence spectrum was read by a fluorescence spectrometer (RF-5301PC, Shimadzu) at an excitation wavelength of 334 nm and emission wavelengths of 373 and 384 nm. The intensity ratio I373/I384 was calculated separately for each concentration, and the point of intersection of the two regression lines was taken to be the estimated CMC. DOX release behavior from cleavable LH-DOX was assessed by incubating the conjugate at different pH (7.4, 6.5 and 5.0) conditions in phosphate buffer at 37 °C using dialysis tubes. At different time intervals, from 0 to 48 h, 100 μL of buffer was collected. The fluorescence spectra of DOX were measured. 2.4. Hemolysis test A hemolysis test was performed as previously described23. Red blood cells were obtained from fresh mouse blood by centrifugation. Then, a 2% blood erythrocyte suspension was incubated with LMWH and LH-DOX at different concentrations. After gently shaking at 37 °C for 1 h, the samples were centrifuged at 3000 rpm (1000 g) for 10 min. Then, the absorbance of the supernatant was measured by a microplate reader at 540 nm, while the precipitated erythrocytes were imaged with a microscope. A hemolysis ratio (%) of less than 5% was regarded as nontoxic. Hemolysis (%) = [A (sample) – A (PBS)]/[A (ultrapure water) – A (PBS)] × 100. PBS and ultrapure water-treated erythrocyte suspensions were used as negative and positive controls, respectively. 2.4. Cellular uptake study of nanoparticles in vitro For cellular uptake analysis, 4 T1 cells were plated in 12-well plates at a density of 1 × 105 cells/well and incubated overnight for attachment. Then, cleavable and non-cleavable LH-DOX nanoparticles were added into the wells after incubating at pH 6.5 or pH 7.4 culture medium for different periods of time. Two hours later, the cells were washed with cold PBS twice, trypsinized, and resuspended in 0.4 mL of PBS. The fluorescence intensity was detected using a flow cytometer (Cytomics FC 500, Beckman Coulter). 2.5. Cytotoxicity study The cytotoxicity assays were performed using MTT. Briefly, B16F10 cells were seeded in 96-well plates at a density of 2 × 105 cells/well with serum-containing culture medium. After incubation overnight, cleavable and noncleavable LH-DOX nanoparticles of different concentrations were added into the wells at pH 6.5 or pH 7.4 for 12 h. Then, the culture medium was replaced with fresh DMEM at pH 7.4 for 24 h, and MTT solution (5 mg/mL) was added to the wells. The medium was replaced 4 h later with 150 μL of dimethyl sulfoxide and the absorbance was measured at 570 nm using a microplate reader. 2.6. Distribution and pharmacokinetic studies Male C57BL/6 mice (20 ± 2 g) were used for the in vivo pharmacokinetic studies. The mice were randomly divided into three groups (n = 6) and intravenously injected 5

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with free DOX, cleavable and non-cleavable LH-DOX at the DOX dose of 5 mg/kg. Blood samples were obtained at 0.5, 1, 2, 4, 8, 24 and 48 h after intravenous administration. For analysis of the tissues samples, each tissue was washed and weighed. Then, 200 µL of the tissue homogenates was mixed with 20 µL of doxorubicin (200 µg/mL) as an internal standard solution. After extraction, the mixture was centrifuged (12,000 rpm, 5 min), and the organic phase was transferred to another centrifuge tube and dried under a stream of nitrogen at 37 °C. The residue was dissolved in 100 µL of the mobile phase solvent. After centrifugation (12,000 rpm, 10 min), the supernatant was collected for HPLC analysis using a UV detector at 254 nm. Similarly, the plasma concentrations of DOX were determined by HPLC analysis. 2.7. In vitro inhibitory effect on cell migration and invasion A wound healing assay was utilized to evaluate the ability of cells to migrate. Briefly, B16F10 cells were cultured to near confluence (90%) in 6-well plates. Scratch wounds were generated with a sterile pipette tip and cells were washed. After treatment with LMWH, DOX or LH-DOX for 24 h, the wound closure was monitored by optical microscopy. Images were captured at 0 h and 24 h. For the transwell invasion assay, 1 × 105 cells were plated in the top chamber pre-coated with 60 μL of Matrigel (24-well insert, pore size: 8 μm) in medium containing 0.5% serum. Medium containing 10% FBS served as a chemoattractant in the lower chamber. After incubation with LMWH, DOX or LH-DOX for 12 h, cells that did not invade through the pores were gently removed with a cotton swab. 2.8. Adherence of platelets to tumor cells Platelet adherence assays were performed as described by Kim et al24. Briefly, B16F10 cells were seeded in 6-well plates and incubated to 50% confluency overnight. Then, the cells were stained with DiD (5 μM) for 30 min. Meanwhile, platelets were obtained and labeled with calcein-AM (5 μM) for 30 min. Subsequently, platelets were added to the stained B16F10 cells in the presence of the antagonist P-selectin, LMWH, or LH-DOX. After incubating for 15 min at 37 °C with gentle shaking, cells were washed twice with PBS. Images were visualized by confocal laser scanning microscopy. 2.9. In vivo interactions of tumor cells with platelets B16F10 cells were labeled with 15 μM CFSE for 15 min at 37 °C with gentle shaking in the dark. Then, 2 × 105 B16F10 cells in 100 μL of PBS were injected into the tail vein of C57BL/6 mice, followed by injection of LMWH, DOX or LH-DOX. Lungs were obtained for analysis after 30 min. Frozen lung sections were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100, followed by incubation with 1% FBS to block nonspecific labeling. Then, the sections were stained with anti-ITGA2B for platelets and DAPI for cell nuclei. After washing with PBS, images were captured using confocal microscopy.

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Molecular Pharmaceutics

2.10. Experimental metastasis assays Mice were intravenously injected directly with 2 × 105 4T1-Lus cells in 100 μL of PBS via the tail vein. Treatment groups were divided into 4 groups: PBS, LMWH, DOX and LH-DOX groups. Intravenous therapy was started with DOX or LH-DOX (DOX-equivalent, 2.5 mg/kg DOX) every three days for a total of 5 doses starting after cell tail vein injection 12 h. Meanwhile, LMWH was intravenously administration every day for 10 days at a dose of 10 mg/kg. Metastatic disease progression in 4T1 tumor-bearing mice was monitored by bioluminescence imaging on days 5, 10 and 20. 2.11. In vivo anti-tumor effect The ability of LH-DOX to inhibit the growth and metastasis of tumors was evaluated. Mice bearing a B16F10 tumors were randomly assigned to 4 groups (n = 7), and the intravenous treatment was started when the tumor reached approximately 100 mm3. Mice were given LH-DOX and DOX (equivalent to 2.5 mg/kg DOX) every two days for a total of five times. LMWH (10 mg/kg) was given every day for 10 days. The body weight and tumor volume were recorded and calculated. On the 24th day, mice were sacrificed. To evaluate the effects of LH-DOX in the later stage of metastasis, 2 × 105 cells suspended in 0.2 mL of sodium chloride aqueous solution were injected directly into the vein tail. Intravenous therapy was started every three days for a total of 5 doses starting at day 1. Mice were sacrificed on day 18. The lung tissues were collected and imaged. 2.12. Preliminary safety assessment To evaluate the in vivo toxicity of LH-DOX, healthy mice weighing 18–22 g were treated with saline, LMWH, or UFH every day for 10 days. DOX and LH-DOX were given (equivalent to 2.5 mg/kg DOX) every three days for a total of 5 doses. Mice weights were measured. Then, 24 h after the last administration, whole blood was obtained for hematology and blood biochemistry was analyzed with a MEK-6318K Automated Hematology Analyzer. In addition, biochemical criteria in serum were analyzed using a Hitachi 7020 automatic biochemical analyzer. 2.13. Statistical analysis Statistical comparisons were performed by analysis of variance (ANOVA) for multiple groups, and p values < 0.05 and < 0.01 were considered to indicate a significant difference and a statistically significant difference, respectively. All data are presented as the mean ±standard deviation. 3. Results 3.1. Synthesis and characterization of LH-DOX Our purpose was to design a safe and efficient amphiphilic fragment comprising the antitumor drug DOX and the anti-metastatic agent LMWH connected via a pH-sensitive linker. In this study, LMWH was considered an ideal carrier because it is a biomacromolecule with hydrophilic and multifunctional activity. Moreover, it is 7

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considered safer than the unfractionated heparin typically used in the clinic. 6-Aminohexanoate hydrochloride was first synthesized via an esterification reaction between a carboxylic acid and ethanol. After hydrazinolysis and the subsequent reaction with DOX, the product was obtained and confirmed by 1HNMR (Supporting information, Fig. S1-S5). The remaining anhydride group was then reacted with different proportions of the carboxy group of LMWH, resulting in a series of cleavable amphiphilic fragments. These conjugates formed self-assembled nanoparticles in aqueous solution, as confirmed by DLS and TEM (Fig. 2A). The TEM images revealed an approximately spherical shape. DLS measurements showed that the mean diameters of conjugates 1, 2, and 3 were 178.1 ± 7.2, 171.7 ±2.8, and 155.2 ±6.6 nm, respectively, and the zeta potential was approximately -25.8 to -33.2 mV (Table 1). Meanwhile, non-cleavable LH-DOX also presented a similar size and zeta potential compared to cleavable LH-DOX (4:1). Conjugates that were under 200 nm were all suitable to accumulate at the tumor site. Additionally, both the cleavable and non-cleavable LH-DOX (4:1) had similar DOX content and a CMC value smaller than that of LH-DOX (1:1). Because of the optimal particle size, size distribution and smaller CMC value, we chose the 4:1 feed ratio of LMWH: DOX and evaluated the stability and drug release properties. The degree of aggregation in serum is an important factor to predict the fate of nanoparticles in vivo25. Diameters of nanoparticles were measured in 50% FBS to test the serum stability. As shown in Fig. 2B, the particle sizes of all the nanoparticles exhibited little change for 48 h. The release experiment was performed at different conditions and is shown in Fig. 2C. Cleavable LD-DOX displayed a pH-sensitive drug release profile with only 30% drug release at pH 7.4 after 48 h. In contrast, over 70% of DOX was released at pH 6.5 and pH 5.0. Table 1. Conjugate

Feed ratio LMWH: DOX (mol)

Size (nm)

PDI

1 (cleavable)

1:1

178.1 ±7.2

0.235

2 (cleavable)

2:1

171.7 ±2.8

3 (cleavable)

4:1

155.2 ±6.6

Zeta potential (mV)

Dox Contenta (%)

CMC (mg/mL)

-25.8 ±4.9

12.98

4.21× 10-2

0.208

-30.7 ±5.2

10.97

4.09× 10-2

0.198

-33.2 ±7.8

9.23

3.52× 10-2

8.87

3.78× 10-2

4 (non-cleavable) 4:1 151.8 ±5.9 0.191 -35.8 ±8.2 LH-DOX) PDI: Polymer dispersion index. CMC: Critical micelle concentration. a: Weight percentage of doxorubicin to heparin nanoparticles Errors are standard errors of the mean (SEM) for N= 3 independent experiments.

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Fig. 2. A. Representative transmission electron microscopy images of nanoparticles (a. C (cleavable) LH-DOX 1:1, b. 2:1, c. 4:1, d. NC (non-cleavable) LH-DOX). The scale bar represents 150 nm. B. Size changes of nanoparticles in 50% FBS for 48 h. C. Drug release profiles of C LH-DOX (4:1) under various pH conditions (pH 5.0, pH 6.5 and pH 7.4) for 48 h. Errors are standard errors of the mean (SEM) for N=3 independent experiments.

3.2. Cellular uptake and MTT assays In the circulating process of these nanoparticles, hydrophilic LMWH could prevent the nanoparticles from interacting with plasma proteins for prolonged circulation. However, it was disadvantage for cellular uptake in turn. An acid-sensitive linker could make the drug DOX effectively separate from nanoparticles in the tumor microenvironment, which could further contribute to the antitumor effect26. To verify this hypothesis, 4T1 cells were treated with nanoparticles in medium at pH 6.5 or pH 7.4 for different lengths of time and then analyzed by flow cytometry. The mean fluorescence intensity of DOX after incubation with the nanoparticles for 2, 4 and 8 h is shown in Fig. 3A. For the non-cleavable group, the cellular uptake level was time-dependent, but it showed little difference at pH 6.5 and pH 7.4. In contrast, cleavable LH-DOX exhibited not only a clear time-dependent internalization but also showed considerably more uptake at pH 6.5 than at pH 7.4. This is likely because the linker was broken in the acidic environment. Hence, the DOX could easily enter the cells via free diffusion27. The cytotoxicity was measured by MTT assays, and the results are summarized in Fig. 3B. Cleavable and non-cleavable LH-DOX exhibited similar enhanced anti-proliferation effects as drug concentrations increased at pH 7.4. There was no obvious difference between the cleavable and non-cleavable LH-DOX groups under normal conditions. However, the cell inhibition of cleavable LH-DOX was clearly higher than that of non-cleavable LH-DOX at pH 6.5 at various concentrations, which was consistent with the cellular uptake observations. This suggested that LMWH 9

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deshielding of cleavable LH-DOX facilitated internalization of DOX, which thus improved the cytocidal effect at pH 6.5.

Fig. 3. A. In vitro cellular uptake of NC LH-DOX and C LH-DOX on 4 T1 cells was measured quantitatively by flow cytometry at diffident pH conditions. B. Cytotoxicity assays of NC LH-DOX and C LH-DOX on 4 T1 cells. (n = 3, mean ± SD, * represents p