Lymphoma immunochemotherapy: targeted delivery of doxorubicin via

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Lymphoma immunochemotherapy: targeted delivery of doxorubicin via a dual functional nanocarrier Qianyu Zhai, Yichao Chen, Jieni Xu, Yixian Huang, Jingjing Sun, Yanhua Liu, Xiaolan Zhang, Song Li, and Suoqin Tang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00606 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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Molecular Pharmaceutics

Lymphoma immunochemotherapy: targeted delivery of doxorubicin via a dual functional nanocarrier †¶§ ‡§ ‡ ‡ ‡ ǁ ‡ Qianyu, Zhai , Yichao Chen , Jieni Xu , Yixian Huang , Jingjing Sun , Yanhua Liu , Xiaolan Zhang , Song ‡ † Li * and Suoqin Tang *.

†

¶ Department of Pediatrics, People's Liberation Army General Hospital, Beijing 100853, China; Department of ‡ Pediatrics, The Third Central Hospital of Tianjin City, Tianjin 300170, China; Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania ǁ 15261, USA. Department of Pharmaceutics, School of Pharmacy, Ningxia Medical University, No. 1160, Shengli § Street, Yinchuan 750004, China. These authors contributed equally to this work. *Correspondence and requests for materials should be addressed to S.L. (email: [email protected]) or S.T. ([email protected]).

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Abstract Chemotherapy drug (paclitaxel, PTX) incorporated in a dual functional polymeric nanocarrier, PEG-Fmoc-NLG, has shown promise as immunochemotherapy in a murine breast cancer model, 4T1.2. The formulation is composed of an amphiphilic polymer with a built-in immunotherapy drug NLG919 that exhibits the immunostimulatory ability through the inhibition of Indoleamine 2,3-dioxygenase 1 (IDO-1) in cancer cells. This work evaluates whether the PEG-derivatized NLG polymer can also be used for delivery of doxorubicin (Dox) in treatment of leukemia. The Dox-loaded micelles were self-assembled from PEG-Fmoc-NLG conjugate, which have a spherical shape with a uniform size of ~120 nm. In cultured murine lymphocytic leukemia cells (A20), Dox-loaded PEG-Fmoc-NLG micelles showed a cytotoxicity that was comparable to that of free Dox. For in vivo studies, significantly improved antitumor activity was observed for Dox/PEG-Fmoc-NLG group compared to Doxil or free Dox group in an A20 lymphoma mouse model. Flow cytometric analysis showed that treatment with Dox/PEG-Fmoc-NLG micelles led to significant increases in the numbers of both total CD4+/CD8+ T cells and the functional CD4+/CD8+ T cells with concomitant decreases in the numbers of myeloid-derived suppressor cells (MDSCs) and Regulatory T cell (Treg) cells. Dox/PEG-Fmoc-NLG may represent a promising immunochemotherapy for lymphoma, which warrants more studies in the future.


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Introduction Doxorubicin (Dox) is an anthracycline antibiotic considered as the most effective chemotherapy drug used for the treatment of cancers including acute lymphoblastic and myelogenous leukemia, breast cancer and bladder cancer1-3. Its works by intercalation into DNA, inhibition of topoisomerase II, production of reactive oxygen species (ROS), induction of p53, and activation of caspases4, 5. Recently, preclinical data indicate that the therapeutic efficacy of Dox is also attributed to the immune mechanisms

6, 7

. Dox was reported to trigger immunogenic cell death by promoting

tumor infiltration of IL-17-secreting γδT cells and enhancing the proliferation and activation of IFNγ-secreting CD8+T cells in tumor draining lymph nodes8-10. Dox can also increase the permeability of tumor cells to granzyme B10. Despite the effectiveness of Dox in cancer treatment, the clinical use of Dox is compromised by its short biological half-life, nonselective in vivo distribution, development of drug resistance and, most seriously, the severe toxicity including cardiomyopathy 11-14.

In order to overcome the side effects of Dox and further enhance its antitumor efficacy, one proven strategy has been to encapsulate the drug in a carrier system that decreases Dox heart distribution and increases targeted delivery to tumors15, 16. Doxil (doxorubicin HCl liposome) is an FDA approved formulation to deliver doxorubicin with decreased cardiotoxicity12. However, Doxil shows limited improvement in therapeutic efficacy over free Dox in clinic, largely due to ineffective release of Dox from Doxil at tumor site17. Recently, several polymer-based delivery systems like polymeric micelles and synthetic polymer conjugates have been designed to deliver



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Dox in vivo, among which polymeric micelles have drawn increasing interests due to their advantages in drug delivery applications18-21. Micelles are usually composed of amphiphilic polymers. The hydrophobic components can form a hydrophobic core to accommodate water-insoluble anti-tumor drugs, and the hydrophilic part such as poly ethylene glycol (PEG) can form a protective corona that stabilizes the micelles in aqueous solution22-25. Micelles usually have small sizes around 20-200 nm, which can facilitate their extravasation at leaky tumor vasculature while avoiding renal clearance and non-specific reticuloendothelial uptake25, 26. Various Dox micellar formulations have been reported that demonstrated improved antitumor activity over free Dox in mouse tumor models19, 27, 28.

We have recently reported an immunostimulatory micellar carrier, PEG-Fmoc-NLG that is based on PEG-derivatized NLG919, a small molecule inhibitor of Indoleamine 2,3-dioxygenase 1 (IDO-1)29. IDO-1 is one of the reported immune checkpoint molecules that include CTLA4, LAG3, PD-1 and TIM3. IDO-1 is an inducible enzyme that catalyzes the tryptophan catabolism30. This enzyme is overexpressed in tumor and causes immunosuppression through depletion of tryptophan and inhibition of effector T cells proliferation30. Studies have reported that IDO inhibition synergizes with several commonly used chemotherapeutic agents such as doxorubicin, cisplatin and cyclophosphamide to elicit significant tumor regression in a tumor-bearing MMTV-Neu mouse model30.


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NLG919 is a potent IDO-1 selective inhibitor with a Ki of 7 nM and EC50 of 75 nM. However, NLG919 has limited solubility in aqueous solutions. PEG modification of NLG919 led to a significant increase in its water solubility. More importantly, PEG-Fmoc-NLG self-assembles to form micelles that are capable of codelivery of other hydrophobic anticancer agents. We previously demonstrated the PTX could be successfully formulated into the PEG-Fmoc-NLG micellar system and in vivo administration of PTX/PEG-Fmoc-NLG micellar formulation led to a significantly improved antitumor response in a breast cancer mouse model29. In this report PEG-Fmoc-NLG was examined for its potential in delivery of Dox for a combined immunochemotherapy in a mouse model of lymphoma. The impact of the combination therapy on the tumor immune microenvironment was also investigated.

Method Cell lines and mice

The A20 B cell lymphoma line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). A20 cells were cultured in complete RPMI-1640 (Sigma, Dorset, UK) medium with 10% FBS, penicillin (50 U/ml) and streptomycin (50 U/ml) at 37°C. Female BALB/c mice of 4–6 weeks were obtained from Charles River Labs. Mice were maintained under pathogen-free conditions according to Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines. All animal-related experiments were performed in full compliance with institutional guidelines at University of Pittsburgh.



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Preparation of blank micelles and Dox/PEG-Fmoc-NLG mixed micelles Dox-loaded polymeric micelles were prepared by a film hydration method. Briefly, Dox·HCl was first mixed with triethylamine (3 equiv.) in DCM/methanol (MeOH) (1:1, v/v). Dox-loaded PEG-Fmoc-NLG micelles were then prepared by mixing PEG-Fmoc-NLG conjugate (10 mM in DCM) and Dox (10 mM in DCM/MeOH) at various carrier/drug ratios. The blank micelles were prepared as previously reported by adding PEG-Fmoc-NLG DCM solution (10 mM) to glass tube29,

31

. Solvent was

removed to form a thin film and the carrier alone or drug/carrier mixture was further dried under vacuum for 1 h. The blank and Dox-loaded micelles were prepared by adding PBS to hydrate the film. Dox loaded into micelles was examined by high performance liquid chromatography (HPLC) with Waters Alliance 2695 Separations Module combined with Waters 2475 Fluorescence Detector (excitation, 490 nm; emission, 590 nm). Hibar 250−4 LiChrosorb RP-8 (5 µm) column was used, and the mobile phase consisted of acetonitrile/water (52.5:47.5, v/v) with 2.5 mM CH3COONH4 and 0.05% (v/v) CH3COOH. The flow rate of the mobile phase was 1 mL/min, and the running time was 12 min. Drug loading capacity (DLC) and drug loading efficiency (DLE) were calculated from the following equations: DLC(%) = [weight of formulated drug/(weight of polymer + drug)] × 100% DLE(%) = (weight of formulated drug/weight of input drug) × 100%

Cytotoxicity of Dox-loaded micelles


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The cytotoxicity of Dox-formulated micelles was examined on A20 cells. Cells were plated in a 96-well plate and then treated with various concentrations of carrier alone, Dox-loaded micelles, Doxil or free Dox. Doxil was prepared in our lab following a published protocol32. The cell viability was evaluated after 72h with MTT assay and cell survival was calculated as the percentage of untreated control group.

Cellular uptake of Dox-loaded micelles

A20 cells were treated with free Dox or Dox-loaded micelles with untreated cells as a control. After 30 min or 2h, cell culture medium was removed and the cells were washed with cold PBS for three times and stained with DAPI for 15min. The uptake of the Dox by A20 cells was observed under a confocal microscope.

In vitro drug release study

In vitro Dox release from the Dox-loaded micelles was examined by a dialysis method. Briefly, 500 µL of free Dox or Dox-loaded micelles were placed in a dialysis bag with a 3.5K MWCO. The dialysis bags were incubated in 100 mL of PBS (pH = 7.4) at 37 °C with gentle shaking. At 0, 1, 2, 4, 8, 12, 24, and 48h, 2 mL of the media were collected and replaced by the same amount of fresh media. The released Dox was quantified using HPLC. The results represented the means of triplicated samples, and all data were expressed as the mean ± SEM.

Plasma Pharmacokinetics of Dox-formulated micelles



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Female BALB/c mice were i.v. injected with Dox·HCl or Dox-formulated PEG-Fmoc-NLG micelles (5 mg Dox/kg) through tail veins. The blood samples were collected in heparinized tubes at different time points (3min, 10min, 30min, 1h, 2h, 4h, 8h, and 12h) post injection. The blood samples were centrifuged at 12,500 rpm for 10 min, and plasma was collected. Dox was extracted by acetonitrile twice and Dox concentrations were examined by HPLC33. PK data were analyzed by WinNonlin using the noncompartmental analysis.

In vivo therapeutic study

The anti-tumor activity of Dox/PEG-Fmoc-NLG micelles was tested in a murine A20 5

lymphoma mouse model using BALB/c mice. In brief, 2 × 10 A20 cells were s.c. injected to the right flank of the mice. When the tumor volume reached 50 mm3, mice were randomly divided to 5 groups and treated intravenously with various treatments every two days for three times. Tumor size was measured every three days starting from the first day of treatment. The tumor volume was calculated according to the equation V = (length of tumor × width of tumor2)/2. Tumor tissues in each group were harvested and weighed at day 21. Hearts were collected and fixed in 4% paraformaldehyde overnight and sectioned to detect cardiac toxicity with H&E staining.

Statistical analysis


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All statistical analyses were carried out using SPSS 15.0 software using one-way analysis of variance (ANOVA). A P value < 0.05 for a 2-tailed test was considered statistically significant.

Results

In vitro characterization of drug-loaded PEG-Fmoc-NLG micelles

PEG-Fmoc-NLG is an amphiphilic molecule, which readily forms small-sized (~100 nm) micelles in aqueous solutions. The drug-loaded micelles were prepared by film hydration method using the hydrophobic drug, Dox. The self-assembly of PEG-Fmoc-NLG and the loading of Dox into the PEG-Fmoc-NLG micelles were tested by dynamic light scattering (DLS) and transmission electron microscopy (TEM) (Figure. 1). Loading of Dox into PEG-Fmoc-NLG micelles resulted in small changes in the sizes of the particles and decreases in PDIs (Figure 1, Table 1). Table 1 shows the sizes, stability, drug loading capacity, and drug loading efficiency of Dox/PEG-Fmoc-NLG at various carrier/drug molar ratios. A minimal carrier/drug molar ratio of 1:1 was needed to formulate the drug in PEG-Fmoc-NLG micelles (Table 1). At this carrier/drug ratio, the Dox-loaded micelles had a mean size of ~95 nm with a small polydispersity index of 0.16. The DLC for Dox/PEG-Fmoc-NLG mixed micelles at this carrier/drug ratio was 15.6% with a high DLE of 93.6%. The Dox-loaded micelles were stable for 12h at room temperature and 7 days at 4 °C. Increasing the carrier/drug ratios led to further increases in the colloidal stability of the Dox-loaded micelles.



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The in vitro release study Figure 2 shows the release of Dox from the various formulations. Free Dox was rapidly diffused across the dialysis bag with more than 60% of Dox being released at the first 4h and 80% of Dox was released from the dialysis bag after 24h. In contrast, Dox showed a more sustained release from Dox/PEG-Fmoc-NLG formulation with less than 20% of Dox released and diffused out of the dialysis bag within 4 h and more than 70% of the Dox remained associated with the micelles after 48h.

In vitro cytotoxicity study

The cytotoxicity of Dox-loaded PEG-Fmoc-NLG micelles was tested in A20 cells (Figure 3). The carrier alone was not effective in killing the tumor cells in vitro at the tested concentrations. Free Dox and Doxil inhibited the tumor cell proliferation in a concentration-dependent manner with an IC50 of 0.70μg/mL and 1.0μg/mL， respectively. Dox-loaded PEG-Fmoc-NLG micelles (IC50=0.58 μ g/mL) were comparable to free Dox in cytotoxicity and showed slightly better tumor cell killing effect than Doxil (Figure 3). Intracellular trafficking Intracellular uptake and distribution of Dox/PEG-Fmoc-NLG micelles were detected by a confocal microscope using Dox as a fluorescence probe. A20 cells were incubated with free Dox or Dox/PEG-Fmoc-NLG micelles for 30 min and 2h, respectively. At 30 min, Dox was found to be distributed largely in cytoplasm for both Dox and Dox/PEG-Fmoc-NLG mixed micelles (Figure 4A). After 2 h, more


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Dox signals in nuclei were found for Dox/PEG-Fmoc-NLG compared to free Dox (Figure 4B), suggesting that the encapsulation of Dox into polymeric micelles enhances the delivery of Dox into cells.

Prolonged blood circulation of Dox formulated in PEG-Fmoc-NLG micelles.

In order to show whether Dox formulated in PEG-Fmoc-NLG micelles could circulate in the blood for a longer period than free Dox, plasma Dox concentrations were evaluated at different time points after i.v. administration of Dox.HCl or Dox-loaded PEG-Fmoc-NLG micelles. As shown in Figure 5, the plasma levels of Dox in the Dox.HCl group declined rapidly and reached a nadir in 30 min, whereas the plasma levels of Dox in Dox/PEG-Fmoc-NLG group declined more slowly. The pharmacokinetic parameters (Vd, CL and T1/2) of Dox.HCl or Dox-loaded PEG-Fmoc-NLG micelles are summarized in Table 2. The half-life of Dox in Dox/PEG-Fmoc-NLG group (T1/2=14.4 h) was significantly longer than that for free Dox group (T1/2=0.85 h) and there was also a significant difference in the volume of distribution between free Dox (Vd=0.49 L/kg) and Dox/PEG-Fmoc-NLG (Vd=0.08 L/kg) groups. The clearance value of Dox-loaded micelles (1.74 ml/h) was remarkably lower than that of free Dox (9.83 ml/h). All these data indicated that the Dox-loaded micelles maintained higher concentrations of Dox in plasma and were cleared much more slowly from the body than free Dox.

In vivo antitumor activity of Dox/PEG-Fmoc-NLG micelles The in vivo antitumor experiment was performed in BALB/c mice bearing A20



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tumors. As shown in Figure 6A, free Dox and Doxil group exhibited significant tumor growth inhibition in vivo in comparison with the control group (p

Lymphoma immunochemotherapy: targeted delivery of doxorubicin via

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