Glutathione-Sensitive Hyaluronic Acid-Mercaptopurine Prodrug

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Glutathione-Sensitive Hyaluronic Acid-Mercaptopurine Prodrug Linked via Carbonyl Vinyl Sulfide: A Robust and CD44-Targeted Nanomedicine for Leukemia Jie Qiu, Ru Cheng, Jian Zhang, Huanli Sun, Chao Deng, Fenghua Meng, and Zhiyuan Zhong Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00846 • Publication Date (Web): 23 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017

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Glutathione-Sensitive Hyaluronic Acid-Mercaptopurine Prodrug Linked via Carbonyl Vinyl Sulfide: A Robust and CD44-Targeted Nanomedicine for Leukemia

Jie Qiu, Ru Cheng*, Jian Zhang, Huanli Sun, Chao Deng, Fenghua Meng, and Zhiyuan Zhong* Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, P. R. China.

Abstract 6-Mercaptopurine (6-MP) is an essential medicine used for treating leukemia in the clinics. 6-MP suffers, however, from poor water solubility, low bioavailability and significant side

effects.

Here,

we

designed

CD44-targeted

glutathione-sensitive

hyaluronic

acid-mercaptopurine prodrug (HA-GS-MP) linked via carbonyl vinyl sulfide for safer and enhanced treatment of acute myeloid leukemia (AML). HA-GS-MP obtained with 50 kDa HA and 6-MP conjugation content of 6.9 wt.% showed excellent water solubility with a hydrodynamic size of ca. 15 nm. Intriguingly, HA-GS-MP was extremely stable, without any drug leakage, under physiological environment while rapidly released 6-MP in response to 10 mM glutathione. HA-GS-MP exhibited obvious targetability and markedly enhanced antitumor effect to OCI/AML-2 human acute myeloid leukemia cells (IC50 = 16.9 µg 6-MP equiv./mL). The pharmacokinetic studies displayed that Cy5-labeled HA-GS-MP had a long circulation time in mice (elimination half-life = 4.37 h). The in vivo fluorescence images demonstrated strong and persistent accumulation of Cy5-labeled HA-GS-MP from 4 to 48 h post injection in the subcutaneous OCI/AML-2 tumor in nude mice. Notably, HA-GS-MP

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while causing little side effects induced significantly enhanced growth inhibition of OCI/AML-2 tumor and better survival rate of OCI/AML-2 tumor-bearing mice as compared to free 6-MP. Carbonyl vinyl sulfide-linked hyaluronic acid-mercaptopurine prodrug has appeared to be a simple and smart nanomedicine for targeted treatment of acute myeloid leukemia. Keywords: 6-Mercaptopurine; prodrug; hyaluronic acid; reduction-sensitive; targeted delivery; leukemia

1. Introduction Leukemia is a malignant blood cancer that causes hundreds of thousands death per year.1 Most leukemia patients are treated with chemotherapy, among which the antimetabolite, 6-mercaptopurine (6-MP), is one of the longest and most used antileukemic drugs in the clinics.2, 3 6-MP has been classified by the World Health Organization as an essential medicine. It takes effect through interfering synthesis of adenine and guanine ribonucleotide, which are important precursors of DNA and RNA. 6-MP suffers, however, from poor water solubility, short plasma half-life, low bioavailability and significant side effects including bone marrow and liver toxicity.4, 5 Different approaches have been employed to achieve enhanced delivery of 6-MP. For instance, Kumar et al. reported that 6-MP-loaded chitosan nanoparticles showed similar proliferative inhibition activity to MCF-7 human breast cancer and HT-1080 fibrosarcoma cells in vitro and better pharmacokinetic profiles in vivo as compared with free 6-MP.6 Glutathione-responsive nanomedicines have attracted recent interest for enhanced cytoplasmic drug release.7-10 Taking advantage of its thiol functional group, 6-MP was conjugated to pyridyldisulfide-functionalized polymers via thiol-disulfide exchange,11-14 to the

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surface of mercapto-modified mesoporous silica nanoparticles (MSNs),15-18 or directly to gold or ZnO nanoparticles.19-22 Notably, due to its low reactivity and low free thiol content, conjugation of 6-MP to polymers via thiol-disulfide exchange yields typically a low drug content.12 To reduce its interaction with serum proteins and increase bioavailability, a couple of

6-MP

derivatives

such

as

cis-3-(9H-purin-6-ylthio)-acrylic

acid

(PTA)

and

cis-6-(2-acetylvinylthio) purine linked by carbonyl vinyl sulfide bond that is cleavable by glutathione (GSH) have been developed as an alternative to 6-MP.23, 24 Interestingly, Zheng et al. reported that PTA-grafted polymeric prodrug micelles had fast intracellular 6-MP release and enhanced cytotoxicity in HL-60 human promyelocytic leukemia cells compared with free 6-MP.25, 26 Here, we designed CD44-targeted glutathione-sensitive hyaluronic acid-mercaptopurine prodrug (HA-GS-MP) linked via carbonyl vinyl sulfide for safer and enhanced treatment of acute myeloid leukemia (AML) (Scheme 1). HA is a biodegradable and biocompatible natural material.27-29 Interestingly, HA has shown a high specific affinity to CD44-overexpressed cancer cells.30-39 Many leukemic cells such as human acute myeloid leukemia cells (AML-1, AML-2), T-cell prolymphocytic leukemia cells, and B-cell chronic lymphocytic leukemia cells were reported to overexpress CD44.40, 41 Notably, Jin et al. reported that targeting of CD44 eradicated human AML stem cells that are responsible for initiating and maintaining the leukemic clonal hierarchy.42 We hypothesized that HA-GS-MP might target to both human leukemic cells and stem cells, leading to effective treatment of acute myeloid leukemia. In this paper, synthesis of HA-GS-MP, its selectivity and antitumor activity to human OCI/AML-2 cancer cells, and therapeutic efficacy toward human OCI/AML-2 tumor-bearing nude mice were investigated.

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Scheme

1.

Illustration

of

GSH-sensitive hyaluronic

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acid-mercaptopurine

prodrug

(HA-GS-MP) linked via carbonyl vinyl sulfide for CD44-targeted treatment of acute myeloid leukemia.

2. Materials and methods 2.1. Synthesis of PTA, HA-Lys and HA-GS-MP PTA was synthesized as reported.23 Briefly, to a stirred solution of 6-MP (182.6 mg, 1.2 mmol) of anhydrous methanol were added sodium methoxide (237.7 mg, 4.4 mmol) and propiolic acid (84.1 mg, 1.2 mmol). The solution was refluxed with continuous stirring overnight and the reaction was quenched by the addition of 4 mL of water. The product was

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precipitated upon adding excess 1 M HCl, filtrated, and purified by re-dissolution in 1 M NaOH and re-precipitation in 1 M HCl. Yield: 80 %. HA-Lys was synthesized in two steps similar to our previous report.27 Firstly, to a stirred solution of HA (497.0 mg, 1.31 mmol carboxyl group) in D.I. water (25 mL) were added H-Lys(Boc)-OMe·HCl (85.9 mg, 0.29 mmol) and DMTMM (127.5 mg, 0.43 mmol). The pH was adjusted to 6.4 - 6.7 by NaOH (1 M). The solution was stirred for 24 h at 35 °C. The resulting HA-Lys(Boc) adduct was isolated by extensive dialysis against water ( MWCO 3500) followed by lyophilization. Yield: 95 %. Secondly, the deprotection of HA-Lys(Boc) using TFA/1 M HCl (v/v 1/1) yielded HA-Lys (DS = 12). HA-GS-MP was synthesized by conjugating PTA to HA-Lys. To a stirred solution of PTA (76.68 mg, 0.35mmol) in anhydrous DMSO (10 mL) was added EDC·HCl (198.64 mg, 1.04mmol) and NHS (39.72 mg, 0.35mmol). The solution was stirred at 30 °C for 3 h to form NHS ester of PTA. A solution of HA-Lys (200 mg, 0.12 mmol amino group) in 5 mL D.I. water was added dropwise and the reaction was stirred at room temperature (r.t.) for 72 h. HA-GS-MP prodrug was acquired by extensive dialysis (MWCO 3500) against DMSO and then D.I. water followed by lyophilization. Yield: 87 %. 1H NMR (DMSO-d6/D2O): HA: δ 2.10, 3.41-4.66; Lys: δ 1.29- 1.69, 2.10, 2.99; PTA: δ 6.54, 8.55, 8.65, 8.99. In a similar way, near-infrared (NIR) fluorophore, cyanine 5 (Cy5)-labeled HA-GS-MP was obtained through conjugating Cy5-NHS ester to HA-GS-MP at r.t.

2.2. Reduction-triggered release of 6-mercaptopurine The release of 6-MP was studied using a dialysis tube (Spectra/Pore, MWCO 12 kDa)

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under 200 rpm shaking at 37 °C in PB (10 mM, pH 7.4) containing 10 % FBS, either in the presence or absence of 10 mM GSH. The release studies were performed at a HA-GS-MP concentration of 0.5 mg/mL and dialyzed 0.5 mL of sample against 25 mL of corresponding release media. At desired time intervals of 0.5, 1, 2, 4, 6, 8, 12 or 24 h, 5 mL of released medium was taken out and replenished with an equal volume of fresh medium. The samples were freeze-dried and the amount of 6-MP was determined by HPLC (Thermo) with UV detection at 308 nm using a mixture of acetonitrile and water (v/v = 5/95) as the mobile phase. Colum: Sepax GP-C18 (150 mm × 4.6 mm, 5 µm), flow rate: 1.0 mL/min; injection volume: 20 µL, retention time: 5.3 min. The data are presented as mean±SD (n = 4).

2.3. Animal models The mice were all handled under protocols approved by Soochow University Laboratory Animal Center and the Animal Care and Use Committee of Soochow University. OCI/AML-2 tumor xenograft model was established by subcutaneous inoculation of 2 × 107 OCI/AML-2 cells in 200 µL of serum free DMEM media into the hind flank of nude mice. Mice with the tumor size of ca. 200-300 mm3 were used for in vivo imaging and biodistribution studies, and mice with tumor size of ca. 50 mm3 were used for therapeutic studies.

2.4. In vivo imaging and blood circulation When the size of tumors reached about 200-300 mm3, 200 µL of Cy5 labeled HA-GS-MP prodrugs was intravenously injected into the tail vein of tumor bearing mice (dosage: 0.5 µg Cy5 equiv./mouse ). The fluorescent scans were performed at various time

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points (2, 4, 8, 12, 24, 36 and 48 h) post i.v. injection using the Maestro in vivo fluorescence imaging system (CRi Inc.) with an excitation band filter at 646 nm and an emission at 670 nm. The inhibitive experiments in vivo was carried out by i.v. injection of free HA solution (50 mg HA/kg) 30 min before administration of Cy5 labeled HA-GS-MP prodrug. In order to study the blood circulation time of HA-GS-MP prodrugs, 150 µL of Cy5-labeled HA-GS-MP or free Cy5 was injected intravenously in Kunming mice (n=3). 10 µL of blood was withdrawn from the orbital at different intervals. Each blood sample was dissolved in 100 µL of 1% Triton X-100 solution followed by sonification. The samples were centrifuged at 10 krpm for 20 min after settling overnight. The supernatant was determined by fluorescence spectrometer after centrifuged at 10 krpm for 20 mins. Its blood circulation follows a two compartment model. The half-lives of two phases (t1 and t2) were calculated according to the following formula:

y = A1 × exp(- x / t1) + A 2 × exp(- x / t 2) + y0

2.5. In vivo antitumor efficacy The in vivo antitumor studies were performed on OCI/AML-2 tumor-bearing mice. When the size of tumor reached 50 mm3, the mice were intravenously injected with HA-GS-MP or free 6-MP at a dosage of 197 µmol 6-MP equiv./kg (corresponding to 30 mg 6-MP equiv./kg) every 2 days for a total of 7 injections. Vernier caliper was used to measure the tumor sizes every 2 days and volume was counted according to the formula V = L × W × W × 0.5, wherein L and W represent the tumor dimension at the longest and widest point, respectively. Relative tumor volumes were calculated as V/V0 (V0 stands for the tumor

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volume when the treatment was initiated). Relative body weights percent were calculated as W/W0×100 (W0 stands for the body weight when the treatment was initiated). Mice were deemed to be dead either when the tumor size increased to 1500 mm3, or when the mice died during treatment.

3. Results and discussion 3.1. Synthesis and Characterization of HA-GS-MP Prodrug HA-GS-MP prodrug was readily synthesized in two steps: (i) addition of 6-MP to propiolic acid to form PTA, and (ii) conjugation of PTA to HA-Lys derivative via carbodiimide chemistry (Scheme 2). It should be noted that there remains a controversy on the optimal molecular weights of HA to achieve the best CD44 targetability.27, 43-45 Here, we selected 50 KDa HA because it has good water solubility as well as prolonged circulation time. It is known that high molecular weight HA has a lower solubility and forms nanoparticles in water by itself while water soluble polymers with a molecular weight higher than 40 KDa is generally required to achieve long circulation time. PTA was obtained with a yield of 80%. 1H NMR (Fig. S1) and elemental analysis showed successful synthesis of PTA. HA-Lys derivative was synthesized with a degree of substitution (DS, number of Lys substituents per 100 sugar units) of 12 as determined by 1H NMR (Fig. S2). PTA was conjugated to HA-Lys at a COOH/NH2 molar ratio of 3/1 in H2O/DMSO (1/2, v/v) using EDC/NHS as a coupling agent for 72 h. The resulting HA-GS-MP conjugate was purified by extensive dialysis. 1H NMR showed signals owing to PTA moieties at δ 6.61, 8.70, and 9.08, and HA-Lys at δ 1.29-1.79, 2.10, 3.28-4.64 (Fig. 1). The comparison of integrals of signals at

8


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δ 6.54 and 8.55 (carbonyl vinyl sulfide protons of PTA) with δ 4.66 (anomeric protons of HA backbone) indicated that 85% of amino groups in HA-Lys was conjugated with PTA, which corresponded to ca. 10.2 6-MP per 100 sugar units and a 6-MP drug content of 6.9 wt.%. UV-Vis measurement at 308 nm showed a drug content of 7.6 wt.%, confirming successful synthesis of HA-GS-MP. Interestingly, HA-GS-MP prodrug was easily dissolved in PB (10 mM). The dynamic light scattering (DLS) showed a small hydrodynamic size of ca. 15 nm (Fig. 2A), indicating that HA-GS-MP prodrug exists as a unimer in PB. Remarkably, the in vitro drug release studies at pH 7.4 and 37 oC revealed that no drug was released in 24 h in the presence of 10% FBS (Fig. 2B), supporting that HA-GS-MP prodrug has excellent stability. In the presence of 10 mM GSH, however, ca. 37.8% and 60.2% of drug was released from HA-GS-MP in 4 and 24 h, respectively. The seemingly saturated drug release is likely related to increasingly smaller difference in drug concentration between inside and outside of the dialysis tube, lower practical GSH concentration (GSH might be oxidized over time) and association of released 6-MP with the proteins in FBS. In comparison, glutathione-sensitive hyaluronic acid-SS-mertansine prodrug showed approximately 12% of drug release in 24 h under physiological condition.35 Trastuzumab DM1 conjugates with disulfide linker were shown not sufficiently stable in clinical trials 46. Hence, glutathione-sensitive carbonyl vinyl sulfide linker appears to have a better stability than disulfide bond under physiological condition. HPLC analyses showed that 6-MP released from the HA-GS-MP prodrug was the same as free 6-MP. These results support that HA-GS-MP prodrug has excellent stability under physiological condition and fast reduction-triggered drug release resulting from the sensitivity of α,β-unsaturated carbonyl bond as reported previously,23 which has a high

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potential to solve the in vivo stability and drug release dilemma encountered by nanomedicines.47, 48

Scheme 2. Synthesis of HA-GS-MP prodrug. Conditions: (i) CH3ONa, CH3OH, 67 ºC, 18 h; (ii) EDC·HCl/NHS, H2O/DMSO, 25 ºC, 72 h.

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Figure 1. 1H NMR spectrum (600 MHz, DMSO-d6/D2O) of HA-GS-MP prodrug.

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A

40 35

Volume (%)

30 25 20 15 10 5 0 1

B

10 Size (nm)

100

100

Cumulative Release (%)

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80

10 mM GSH 60 40 20

No GSH 0 0

5

10 15 Time (h)

20

25

Figure 2. (A) Hydrodynamic size of HA-GS-MP determined by DLS. (B) In vitro drug release behavior of HA-GS-MP prodrug at pH 7.4 and 37 °C in PB (10 mM, pH 7.4) containing 10 %

FBS, either in the presence or absence of 10 mM GSH.

3.2. In vitro Antitumor Effect and CD44-Targetability of HA-GS-MP Prodrug. The in vitro antitumor effect of HA-GS-MP prodrug was evaluated by MTT assays in

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CD44 positive OCI/AML-2 cancer cells. Interestingly, the results showed that HA-GS-MP induced significantly better antitumor effect than free 6-MP, signifying that HA-GS-MP can be efficiently taken up by OCI/AML-2 cells and quickly release 6-MP (Fig. 3). For example, OCI/AML-2 cells treated with HA-GS-MP and 6-MP at a dosage of 30 µg 6-MP equiv./mL had a cell viability of about 36.3 % and 69.4 %, respectively. In contrast, PTA exhibited very low cytotoxicity compared with 6-MP (Fig. 3), likely due to its negative charge under physiological condition that hinders cellular interaction and internalization. In comparison, polymeric 6-MP prodrugs (based on e.g. PEG and chitosan) linked with α,β-unsaturated carbonyl group were reported to cause similar or lower cytotoxicity against Burkitt lymphoma Raji cells, human promyelocytic leukemia HL-60 cells, human breast cancer MCF-7 cells and fibrosarcoma HT-1080 cells.6,

12, 26

The increased cytotoxicity of HA-GS-MP against

OCI/AML-2 cells could be explained by the improved cellular uptake of HA-GS-MP via CD44-mediated endocytosis and fast cytoplasmic drug release.27,

35

In order to assess its

targetability, HA-GS-MP was labeled with Cy5, a near-infrared (NIR) fluorophore, using DMTMM as a coupling agent. UV-vis revealed on average one Cy5 molecule per HA-GS-MP. Flow cytometry demonstrated efficient and specific uptake of HA-GS-MP by OCI/AML-2 cells (Fig. 4). The cellular uptake was significantly inhibited by pretreating OCI/AML-2 cells with free HA, confirming HA-GS-MP is internalized by receptor-mediated manner.

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PTA 6-MP HA-GS-MP

120

***

Cell Viability (%)

100

**

80

*** ***

60 40 20 0 5

10 20 6-MP Conc. (µg/mL)

30

Figure 3. In vitro cytotoxicity of 6-MP, PTA and HA-GS-MP in OCI/AML-2 cells at 48 h incubation (n=4).

PBS Free HA + HA-GS-MP HA-GS-MP

80 70 60 Counts

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50 40 30 20 10 0 1

10

100 1000 Fluorescence Intensity

10000

Figure 4. Flow cytometry of OCI/AML-2 cells after 2 h cultivation with Cy5-labeled HA-GS-MP. The inhibitive experiments were conducted by 4 h pretreatment of OCI/AML-2 cells with 5 mg/mL of

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free HA before adding HA-GS-MP prodrug.

3.3. In Vivo Imaging and Pharmacokinetics of HA-GS-MP Prodrug in Tumor Bearing Mice The in vivo fluorescence images of OCI/AML-2 tumor-bearing nude mice displayed significant tumor accumulation of HA-GS-MP prodrug at 4 h post i.v. injection (Fig. 5A). The tumor Cy5 fluorescence reached the maximum at 24 h and kept strong in 36 h, signifying superior tumor-targetability of HA-GS-MP prodrug, as also reported for varying HA-functionalized nanomedicines in different CD44 tumor models.49-51 To assess the role of HA receptors in vivo, tumor-bearing mice were pre-treated with free HA at a dosage of 50 mg/kg prior to injection of Cy5-labeled prodrug. The results showed that tumor accumulation of HA-GS-MP prodrug was significantly weakened by free HA, signifying the importance of ligand-receptor interaction for HA-GS-MP prodrug in achieving high targeting ability and accumulation in the OCI/AML-2 tumor in vivo. The in vivo pharmacokinetic studies showed that Cy5-labeled HA-GS-MP prodrug had a long elimination half-life of 4.37 h in mice (Fig. 5B), which is much longer than that of 6-MP (0.5-1.5 h).52 In sharp contrast, free Cy5 exhibited a short elimination half-life of 0.01 h.

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B

20 Cy5-labeled HA-GS-MP Free Cy5 15 Cy5 Uptake (% ID/g)

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10

5

0 0

5

10 15 Time (h)

20

25

Figure 5. (A) In vivo fluorescence images of HA-GS-MP prodrug in OCI/AML-2 tumor-bearing mice at different time points following intravenous injection of Cy5-labed HA-GS-MP (dosage: 0.5 µg Cy5 equiv./mouse). The control group was conducted by pretreating free HA (50 mg/kg) prior to injection of Cy5-labeled HA-GS-MP. The mouse autofluorescence was removed by spectral unmixing using the Maestro software. (B) In vivo pharmacokinetics of Cy5-labeled HA-GS-MP and free Cy5 in Balb/C mice (dosage: 0.4 µg Cy5 equiv./mouse). Cy5 uptake (%ID/g) is expressed as mean ± SD (n = 3).

3.4. Antitumor Efficacy of HA-GS-MP Prodrug in Vivo To investigate the therapeutic efficacy of HA-GS-MP, human OCI/AML-2 tumor-bearing mice were intravenously injected with HA-GS-MP at 197 µmol 6-MP equiv./kg every two days for 12 days. 6-MP and PBS were used as controls. As expected, saline-treated group showed fast tumor growth inhibition. HA-GS-MP afforded significantly better tumor growth

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inhibition than 6-MP (Fig. 6A). The photographs of tumors isolated on day 20 corroborated that mice treated with HA-GS-MP had the smallest tumor size (Fig. 6B). Notably, both HA-GS-MP and 6-MP did not cause change of body weights (Fig. 6C), indicating that HA-GS-MP does not induce obvious side effects. Interestingly, HA-GS-MP significantly increased the mice survival rate as compared to free 6-MP and PBS groups (median survival time: 56 days versus 32 and 26 days, respectively) (p < 0.05) (Fig. 6D). The histological analysis revealed no significant damage to the major organs of three groups (Fig. 7), while HA-GS-MP treatment caused more extensive damage in the tumor tissue compared with free 6-MP and PBS groups. These results demonstrate that HA-GS-MP brings about significantly better treatment of OCI/AML-2 tumor-bearing mice than free 6-MP. The enhanced antitumor effect of HA-GS-MP is most likely due to its good targetability, high stability during circulation, as well as GSH-triggered fast 6-MP release in the cytosol. Given the fact that both leukemic cells and leukemic stem cells overexpress CD44,35, 40, 42 HA-GS-MP would probably induce significantly more potent treatment of leukemic cancer patients than 6-MP. Moreover, in contrast to prodrugs linked via disulfide bonds that exhibit certain drug leakage,35, 53-56 HA-GS-MP linked via carbonyl vinyl sulfide shows superior stability with zero drug release under physiological condition. The easy synthesis, high stability, CD44-targetability and fast intracellular drug release of HA-GS-MP renders it a highly promising alternative to 6-MP for leukemia treatment.

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Figure 6. In vivo antitumor performance of HA-GS-MP prodrug and free 6-MP in OCI/AML-2 tumor-bearing nude mice. The drugs were given on day 0, 2, 4, 6, 8, 10 and 12 (dosage: 197 µmol 6-MP equiv./kg). (A) Relative tumor volume changes. Data are presented as mean ± SD (n = 5). *p

Glutathione-Sensitive Hyaluronic Acid-Mercaptopurine Prodrug

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