Lymphoma immunochemotherapy: targeted delivery of doxorubicin via

Aug 29, 2017 - ... drug NLG919 that exhibits the immunostimulatory ability through the inhibition of Indoleamine 2,3-dioxygenase 1 (IDO-1) in cancer c...
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Article Cite This: Mol. Pharmaceutics 2017, 14, 3888-3895

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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*,† †

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 15261, United States ∥ Department of Pharmaceutics, School of Pharmacy, Ningxia Medical University, No. 1160, Shengli Street, Yinchuan 750004, China ¶

ABSTRACT: Chemotherapy drug (paclitaxel, PTX) incorporated in a dual functional polymeric nanocarrier, PEG-Fmoc-NLG, has shown promise as an 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 PEGderivatized NLG polymer can also be used for delivery of doxorubicin (Dox) in treatment of leukemia. The Dox-loaded micelles were selfassembled 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 the Dox/PEG-Fmoc-NLG group compared to Doxil or the 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 cells (T reg ). Dox/PEG-Fmoc-NLG may represent a promising immunochemotherapy for lymphoma, which warrants more studies in the future. KEYWORDS: doxorubicin, immunochemotherapy, lymphoma, dual-functional nanocarrier

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INTRODUCTION

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 tumors.15,16 Doxil (doxorubicin HCl liposome) is an FDA approved formulation to deliver doxorubicin with decreased cardiotoxicity.12 However, Doxil shows limited improvement in therapeutic efficacy over free Dox in clinic, largely due to ineffective release of Dox from Doxil at the tumor site.17 Recently, several polymer-based delivery systems like polymeric micelles and synthetic polymer conjugates have been designed to deliver Dox in vivo, among which polymeric micelles have drawn increasing interests due to their advantages in drug delivery applications.18−21 Micelles are usually composed of amphiphilic polymers. The hydrophobic components can form

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 cancer.1−3 Its works by intercalation into DNA, inhibition of topoisomerase II, production of reactive oxygen species (ROS), induction of p53, and activation of caspases.4,5 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 IL17-secreting γδT cells and enhancing the proliferation and activation of IFNγ-secreting CD8+T cells in tumor draining lymph nodes.8−10 Dox can also increase the permeability of tumor cells to granzyme B.10 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 © 2017 American Chemical Society

Received: Revised: Accepted: Published: 3888

July 14, 2017 August 17, 2017 August 29, 2017 August 29, 2017 DOI: 10.1021/acs.molpharmaceut.7b00606 Mol. Pharmaceutics 2017, 14, 3888−3895

Article

Molecular Pharmaceutics

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 a Waters Alliance 2695 Separations Module combined with a Waters 2475 Fluorescence Detector (excitation, 490 nm; emission, 590 nm). A 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:

a hydrophobic core to accommodate water-insoluble antitumor drugs, and the hydrophilic part such as poly ethylene glycol (PEG) can form a protective corona that stabilizes the micelles in aqueous solution.22−25 Micelles usually have small sizes around 20−200 nm, which can facilitate their extravasation at leaky tumor vasculature while avoiding renal clearance and nonspecific reticuloendothelial uptake.25,26 Various Dox micellar formulations have been reported that demonstrated improved antitumor activity over free Dox in mouse tumor models.19,27,28 We have recently reported an immunostimulatory micellar carrier, PEG-Fmoc-NLG, that is based on PEG-derivatized NLG919, a small molecule inhibitor of indoleamine 2,3dioxygenase 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 catabolism.30 This enzyme is overexpressed in tumor and causes immunosuppression through depletion of tryptophan and inhibition of effector T cell proliferation.30 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 model.30 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 model.29 In this article, PEGFmoc-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.

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. 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 protocol.32 The cell viability was evaluated after 72 h 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 2 h, cell culture medium was removed, and the cells were washed with cold PBS for three times and stained with DAPI for 15 min. 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 48 h, 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. 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 (3 min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 12 h) 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 HPLC.33 PK data were analyzed by WinNonlin using the noncompartmental analysis. In Vivo Therapeutic Study. The antitumor activity of Dox/PEG-Fmoc-NLG micelles was tested in a murine A20 lymphoma mouse model using BALB/c mice. In brief, 2 × 105 A20 cells were s.c. injected to the right flank of the mice. When the tumor volume reached 50 mm3, mice were randomly divided into five groups and treated intravenously with various treatments every 2 days for three times. Tumor size was

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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 RPMI1640 (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 Laboratories. 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. Preparation of Blank Micelles and Dox/PEG-FmocNLG 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 tube.29,31 Solvent was removed to form a thin 3889

DOI: 10.1021/acs.molpharmaceut.7b00606 Mol. Pharmaceutics 2017, 14, 3888−3895

Article

Molecular Pharmaceutics

Figure 1. TEM image of the PEG-Fmoc-NLG blank micelles (A) and Dox/PEG-Fmoc-NLG micelles (B). Size distribution of the PEG-Fmoc-NLG blank micelles (C) and Dox/PEG-Fmoc-NLG micelles (D).

measured every 3 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. 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 two-tailed test was considered statistically significant.

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 12 h 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. In Vitro Release Study. Figure 2 shows the release of Dox from the various formulations. Free Dox was rapidly diffused

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RESULTS In Vitro Characterization of Drug-Loaded PEG-FmocNLG 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 selfassembly 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

Figure 2. In vitro release profile of Dox formulated in PEG-Fmoc-NLG micelles.

across the dialysis bag with more than 60% of Dox being released at the first 4 h, and 80% of Dox was released from the dialysis bag after 24 h. 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 48 h. In Vitro Cytotoxicity Study. The cytotoxicity of Doxloaded 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 concentrationdependent manner with an IC50 of 0.70 and 1.0 μg/mL,

Table 1. Biophysical Characterization of Blank Micelles and Dox-Formulated Micelles Micelle PEGFmocNLG Dox/ PEGFmocNLG

molar ratio

1:1 2.5:1 5:1

size (nm)

PDI

106.9

0.338

94.70 117.7 119.7

0.156 0.148 0.147

stability (25 °C)

stability (4 °C)

DLC (%)

DLE (%)

12 h 72 h 96 h

7d 21 d 50 d

15.6 7.12 3.79

93.6 96.0 98.4 3890

DOI: 10.1021/acs.molpharmaceut.7b00606 Mol. Pharmaceutics 2017, 14, 3888−3895

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Molecular Pharmaceutics

micelles for 30 min and 2 h, respectively. At 30 min, Dox was found to be distributed largely in the cytoplasm for both Dox and Dox/PEG-Fmoc-NLG mixed micelles (Figure 4A). After 2 h, more Dox signals in nuclei were found for Dox/PEG-FmocNLG 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-FmocNLG micelles are summarized in Table 2. The half-life of Dox in Dox/PEG-Fmoc-NLG group (T 1/2 = 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

Figure 3. Cytotoxicity of Dox-loaded PEG-Fmoc-NLG micelles in comparison to free Dox and Doxil formulation in A20 murine lymphoma cell line. Cells were treated for 72 h, and cytotoxicity was determined by MTT assay.

respectively. Dox-loaded PEG-Fmoc-NLG micelles (IC50 = 0.58 μg/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

Figure 4. Confocal images showing the intracellular uptake of Dox from free Dox or Dox-loaded PEG-Fmoc-NLG micelles in A20 cells after 30 min and 2 h incubation. 3891

DOI: 10.1021/acs.molpharmaceut.7b00606 Mol. Pharmaceutics 2017, 14, 3888−3895

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Molecular Pharmaceutics

significantly more antitumor effects compared to free Dox or Doxil group (p < 0.01, vs free Dox or Doxil). Dox/PEG-FmocNLG mixed micelles were the most effective in inhibiting the tumor growth (p < 0.01, vs control or free Dox; P < 0.05, vs Doxil or PEG-Fmoc-NLG). At the end of the therapeutic study, tumor tissues were harvested and weighted. The Dox/PEGFmoc-NLG showed the lowest tumor weight with an IR of 87.2% (Figure 6C). The tumor weight order is as follows: Dox/ PEG-Fmoc-NLG < PEG-Fmoc-NLG < Doxil < free Dox < control, which was consistent with the tumor volume measurement (Figure 6A). The mouse body weight change is an important parameter to evaluate formulation toxicity. As shown in Figure 6B, no obvious body weight loss was observed for all of the treatment groups. Histological analysis of heart tissues was carried out to further evaluate the potential toxicity of different treatments. As shown in Figure 7, obvious myocytolysis and myofibrillolysis with fibrils dearrangement were observed in the heart tissue section in the group treated with free Dox. This indicated that free Dox showed cardiac toxicity. In contrast, no significant pathological changes were observed in the heart tissue section in the group treated with carrier alone, Doxil, or Dox-loaded micelles. Assessment of Tumor Infiltrating Lymphocytes. To delineate a role of immune response in the enhanced antitumor activity by the Dox-loaded micelles, we examined the immune cell infiltration in the tumor tissues with various treatments by flow cytometry 1 day following three times of treatments. Figure 8A,C shows that there were more infiltrated CD4+ T cells and CD8+ T cells in the tumors treated with Dox/PEGFmoc-NLG compared to other Dox groups (p < 0.05, vs free Dox, Doxil, or control). Figure 8B,D also showed that there were more IFN-γ+ CD4+ T and IFN-γ+ CD8+ T cells in the tumors treated with Dox/PEG-Fmoc-NLG compared with other groups (p < 0.05, vs control, PEG-Fmoc-NLG, free Dox, or Doxil). The tumors treated with carrier alone also showed enhanced infiltration of total T cells and IFN-γ+ T cells in tumor tissues (p < 0.05, vs control). The numbers of granzyme B-positive CD8+ T cells were also significantly increased in all of the treatment groups compared to the control group (p < 0.05, vs control) (Figure 8E), among which tumors treated with Dox/PEG-Fmoc-NLG and carrier alone showed the highest percentages of granzyme B-positive CD8+ T cells compared to other treatment groups (p < 0.05, vs free Dox or Doxil). Treg cells are known to be immunosuppressive, and they were significantly decreased in tumors treated with free Dox, PEGFmoc-NLG, and Dox/PEG-Fmoc-NLG micelles compared with control group, especially in the tumors treated with Dox/PEG-Fmoc-NLG micelles (p < 0.05, vs control) (Figure 8F). Myeloid-derived suppressor cells (MDSCs) are another major component of the tumor microenvironment. The main feature of these cells is their potent immune suppressive activity. As shown in Figure 8G,H, both granulocytic MDSC (G-MDSC) and monocytic MDSC (M-MDSC) were significantly decreased in the tumors treated with PEG-Fmoc-NLG alone and Dox/PEG-Fmoc-NLG micelles (p < 0.05, vs control, free Dox, or Doxil). We also examined the macrophage infiltration in tumor tissues. Figure 8I,J shows that the numbers of M2 (CD11b+F4/ 80+CD206+) tumor-associated macrophages were significantly reduced in the tumors treated with PEG-Fmoc-NLG and Dox/ PEG-Fmoc-NLG micelles. Meanwhile, the numbers of M1

Figure 5. Pharmacokinetics of Dox in blood in A20 tumor-bearing mice receiving intravenous administration of free Dox or Dox/PEGFmoc-NLG micelles at a Dox dose of 5 mg/kg. Values reported are the means ± SD for triplicate samples.

Table 2. Pharmacokinetic Variables of Free Dox and DoxLoaded PEG-Fmoc-NLG Micelles groups Dox Dox/PEGFmoc-NLG

T1/2 (h)

AUCo‑inf (μg × h/mL)

Cmax (μg/mL)

CL (mL/h)

Vd (L/kg)

0.85 14.4

9.36 54.1

10.9 33.28

9.83 1.74

0.49 0.08

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 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 < 0.05). Interestingly, the PEG-Fmoc-NLG carrier alone showed

Figure 6. In vivo antitumor activity of various Dox formulations in an A20 lymphoma mouse model. Black arrows indicate the i.v. injection. (A) Relative tumor volume. *P < 0.01, compared to control (saline), # P < 0.01, compared to free Dox; &P < 0.05, compared to Doxil, ΦP < 0.05, compared to PEG-Fmoc-NLG. (B) Body weights of different treatment groups. (C) Tumor weight, *P < 0.01, compared to control (saline), #P < 0.01, compared to free Dox, &P < 0.05, compared to Doxil, ΦP < 0.05, compared to PEG-Fmoc-NLG. 3892

DOI: 10.1021/acs.molpharmaceut.7b00606 Mol. Pharmaceutics 2017, 14, 3888−3895

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Molecular Pharmaceutics

Figure 7. H&E staining of heart tissue with various treatments.

Figure 8. Flow cytometry analysis of immune cell subsets in tumor tissues. (a−d) T-cell infiltration in mouse tumors treated with Dox, Doxil, PEGFmoc-NLG or Dox/PEG-Fmoc-NLG at a Dox dosage of 5 mg kg−1. The abundance of CD4+ (A), IFN-positive CD4+ T cells (B), CD8+ (C), IFN-g positive CD8+ T cells (D), and granzyme B-positive CD8+ T cells (E) in tumor tissues were detected by flow cytometry. (F) Flow cytometry analysis of FoxP3+ T regulatory cells in mouse tumors. (G,H) Flow cytometry analysis of Gr-1highCD11b+ granulocytic (G-MDSC) (G) and Gr-1intCD11b+ monocytic (M-MDSC) (H) MDSC subsets. (I) M1-type TAMs (CD11b+/F4/80+/CD206−) and (J) M2-type TAMs (CD11b+/F4/80+/ CD206+) in tumor tissues were detected by flow cytometry. The bars represent means ± SEM (*p < 0.05, vs control group, &p < 0.05, vs PEG-Fmoc-NLG group, Ψp < 0.05, vs free Dox group, δp < 0.05, vs Doxil group, N = 5).

(CD11b +F4/80+CD206−) tumor-associated macrophages were increased in all treatment groups (p < 0.05, vs control, free Dox, or Doxil).

in a breast cancer mouse model, which was due to their preferential tumor accumulation and a possible synergistic effect between NLG and loaded PTX. In this study, we have shown that Dox could also be successfully loaded into PEGFmoc-NLG micelles, resulting in a further improvement in the therapeutic activity compared to Doxil formulation in a lymphoma mouse model (Figure 6). Dox could be readily

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DISCUSSION We have previously reported that PTX-loaded PEG-FmocNLG micelles showed significantly improved antitumor effect 3893

DOI: 10.1021/acs.molpharmaceut.7b00606 Mol. Pharmaceutics 2017, 14, 3888−3895

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Molecular Pharmaceutics

underlying mechanism for the differences in the immune microenvironment between Dox/PEG-Fmoc-NLG- and PTX/ PEG-Fmoc-NLG-treated tumors is not clear and may reflect the different immune modulating activities of Dox and PTX, and/or the different tumor models used in the two different studies (breast cancer vs lymphoma). More studies are needed to better understand the impact of different immunochemotherapies on the tumor microenvironment and the overall antitumor activity. It also remains to be tested if Dox/PEGFmoc-NLG can be combined with other immune checkpoint inhibitors to further improve the outcome of treatment. Recently, an immune checkpoint blockade with antibodies to CTLA4, PD-L1, and PD1 has led to durable response in the clinic; however, the effect was limited to a small population of patients. Identification of potential resistance mechanisms and development of therapeutic strategies is in urgent need. It has been reported that IDO is a critical resistance mechanism in CTLA-4 immunotherapy; the blockade of IDO in the mice treated with anti-CTLA-4 antibody led to significant tumor growth inhibition and prolonged survival time compared to single therapy.35 Similar findings were also observed with antibodies targeting PD-1, PD-L1, and GITR.36 So, combination of the PEG-Fmoc-NLG micelle system and antibodies targeted to other immune checkpoints may provide a new therapeutic regimen to further improve the anticancer efficacy. In summary, Dox could be effectively loaded into PEGFmoc-NLG micelles. The Dox/PEG-Fmoc-NLG micelles had a size around 100 nm and exhibited a sustained drug release profile. In vivo delivery of Dox via PEG-Fmoc-NLG micelles led to an improved tumor immune microenvironment and significantly enhanced antitumor activity in a murine model of lymphoma. Dox/PEG-Fmoc-NLG micelles may represent a new type of immunochemotherapy to improve the treatment of lymphoma.

formulated into PEG-Fmoc-NLG micelles with relatively small sizes (