Enzyme responsiveness enhances the specificity and effectiveness of nanoparticles for the treatment of B16F10 melanoma

Journal Pre-proof Enzyme Responsiveness Enhances the Specificity and Effectiveness of Nanoparticles for the Treatment of B16F10 Melanoma Yi Huang, Yanbin Shi, Qingjie Wang, Tongtong Qi, Xianglei Fu, Zili Gu, Yuhua Zhang, Guangxi Zhai, Xiaogang Zhao, Qifeng Sun, Guimei Lin

PII:

S0168-3659(19)30616-9

DOI:

https://doi.org/10.1016/j.jconrel.2019.10.052

Reference:

COREL 10001

To appear in: Received Date:

15 August 2019

Revised Date:

26 October 2019

Accepted Date:

29 October 2019

Please cite this article as: Huang Y, Shi Y, Wang Q, Qi T, Fu X, Gu Z, Zhang Y, Zhai G, Zhao X, Sun Q, Lin G, Enzyme Responsiveness Enhances the Specificity and Effectiveness of Nanoparticles for the Treatment of B16F10 Melanoma, Journal of Controlled Release (2019), doi: https://doi.org/10.1016/j.jconrel.2019.10.052

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Enzyme Responsiveness Enhances the Specificity and Effectiveness of Nanoparticles for the Treatment of B16F10 Melanoma

Yi Huang1, Yanbin Shi2, Qingjie Wang3,4, Tongtong Qi1, Xianglei Fu1, Zili Gu1, Yuhua Zhang2, Guangxi Zhai1, Xiaogang Zhao5, Qifeng Sun5, Guimei Lin*1

School of Pharmaceutical Science, Shandong University, 44 Wenhuaxi Road, Jinan 250012, PR

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1

China 2

School of Mechanical & Automotive Engineering, Qilu University of Technology (Shandong

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Academy of Sciences), Jinan 250353, China

Institute of Basic Medical Sciences, Qilu Hospital, Shandong University, Jinan 250012, China

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The Key Laboratory of Cardiovasular Remodelling and Function Research, Chinese Ministry of

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Health, Qilu Hospital, Shandong University, Jinan 250012, China Department of Thoracic Surgery, Second Hospital of Shandong University, Jinan 250012, PR

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5

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China

AUTHOR INFORMATION

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Corresponding Authors

*E-mail: [email protected]; phone:

*

Corresponding author. School of Pharmaceutical Science, Shandong University, 44 Wenhuaxi Road, Jinan 250012, PR China.E-mail address: [email protected] (G. Lin).

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Graphic Abstract Under the dual action of the degradation of heparanase-1 and the ion diffusion of the electrolyte in the tumor microenvironment, the particle size reduction and charge turnover of PTX-DOTAP@Alloferon-1-Heparin/Protamine facilitate nanoparticles access to the depth of the tumor. At the same time, Alloferon-1 is released to activate NK cells, which combined with PTX

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to form a dual treatment system of immunochemotherapy.

Highlights: 

High expression of heparanase-1 in the tumor microenvironment was verified in this work. Based on this feature, we have prepared novel and sensitive environmentally responsive nanoparticles.

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The reduction of the particle size and the ζ-potential inversion of the nanoparticles during the depolymerization of the polyelectrolyte complex, which facilitate the more delivery of nanoparticles to the depth of the tumor and more nanoparticles uptake by tumor cells with high negative charges on the surface. 2

The nanoparticles are very smart, and its procedural release function allows PTX and alloferon-1 to be released step by step to act on different target cells, which is equivalent to a nano-scale controlled release formulation.

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In the nanoparticle design process, the toxicity of the carrier material is greatly reduced, which provides a favorable basis for clinical research.

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The almost no organic solvent, uncomplicated preparation process, and uniform particle size make the nanoparticles druggable.

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It is also proved that the degradation process of the nanoparticles at the tumor is the result of the joint action of heparanase-1 and ion diffusion.

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Finally, in vitro and in vivo experiments have demonstrated that the nanoparticles which combine chemotherapy and immunotherapy have excellent therapeutic effects on melanoma.

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

ABSTRACT

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The clinical treatment of melanoma continues to present many challenges including poor

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prognosis because neither monotherapy nor combination therapies have shown maximal treatment

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efficacy. In this study, an enzyme-responsive nanoparticle was designed for tumor subtypes with the high expression of heparanase-1, since highly metastatic tumors such as melanoma generally

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express significant levels of heparanase-1. PTX-DOTAP@alloferon-1-heparin/protamine, an enzyme-responsive nanoparticle, has a particle size of 106.1 ± 1.113 nm and a ζ-potential of -45.1

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± 0.455 mV, which enables enrichment in the tumor site by passive targeting. Subsequently, heparanase-1, which is highly expressed in the extracellular matrix, rapidly recognizes and

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degrades heparin in the outer layer of the nanoparticle and releases encapsulated alloferon-1 by ion diffusion to activate inhibited NK cells in the tumor microenvironment. The size of the smart nanoparticle will eventually decrease to 59.30 ± 0.783 nm and the ζ-potential will reverse to 25.4 ± 0.257 mV, which is beneficial for deep penetration and tumor cell uptake (due to the high negative charge on the tumor cell surface) of PTX-DOTAP cores. Paclitaxel is released in the 3

cytoplasm, and the tumor cells are arrested in the G2/M phase. The nanoparticle characterization experiment demonstrated that in vivo drug delivery could be completed. In subsequent cell and animal experiments, the experimental data demonstrated the efficient therapeutic effects of the nanoparticle. This study provides an excellent template nanoparticle for the treatment of highly metastatic tumors to enhance future prognosis.

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KEYWORDS: Enzyme responsiveness, combined immunochemotherapy, size shrinking, charge turnover

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INTRODUCTION

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Melanoma, originating from melanocytes, is the most complicated of all skin cancers (1).

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Metastatic melanoma reduces the five-year survival rate from 98% to 17% (2). The causes of melanoma in different races are not completely consistent, so the corresponding measures taken in

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early prevention and examination also differ (3). A previous report updated and refined the melanoma staging method, which is conducive to achieving a more rational and effective clinical

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treatment plan to improve drug efficacy and reduce side effects (4). Some researchers have summarized the changes in melanoma treatment programs during the past five years and proposed

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a reasonable front-line treatment option for patients with BRAF-mutated melanoma (5). In short, the current clinical methods of treating melanoma have significantly improved and include chemotherapy, molecular target therapy, immunological checkpoint blockade therapy, and tumor vaccine therapy (2). However, even with the unremitting efforts of research teams from around the world, the poor 4

prognosis of clinical melanoma treatment remains frustrating (6). Several major factors that contribute to poor prognosis include drug resistance, anti-apoptosis, tumor metastasis, and immunosuppression (7). The current effective solution to poor prognosis is stereoscopic response strategy. According to this theory, the current study uses chemotherapy-based (PTX), immune activation-assisted (alloferon-1) stereoscopic response strategy for the treatment of melanoma. As a classic broad-spectrum anticancer drug, paclitaxel inhibits tumor cell proliferation and is

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commonly used for the clinical treatment of melanoma (8). However, the long-term use of

paclitaxel will increase a patient's resistance to it, and the mechanism of drug resistance often

varies from person to person (9). Fortunately, in recent years, the efficacy of immunotherapy has

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become increasingly better for cancer treatment. Compared with traditional single administration,

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researchers have used immunotherapeutics in combination with chemotherapeutic drugs and have

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found that this treatment regimen significantly improved drug efficacy. Alloferon-1, HGVSGHGQHG VHG, a 13 amino acid basic peptide, has a strong positive charge when it is

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dissolved in water. The peptide was first used in the antiviral and antibacterial fields, and has been recently found to have good effects on cancer treatment. Some reports have demonstrated that its

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mechanism for cancer treatment is not the direct killing of tumor cells but rather activating natural killer (NK) cells in the tumor microenvironment. NK cells release interferon-γ (IFN-γ) and tumor

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necrosis factor-α (TNF-α) to reverse tumor cell-mediated immune system inhibition (10). To further achieve an effective stereoscopic response strategy in vivo, the two drugs cannot be delivered separately. Co-delivery should be used to make the pharmacokinetic properties and drug distribution consistent. This facilitates the synchronization of the effects of the two drugs on the tumor site to achieve the best stereoscopic response. Codelivery can solve the problems of 5

insufficient tumor selectivity and tumor accumulation, unpredictable drug/protein ratios at tumor sites, short half-lives, and serious systemic adverse effects (11-13). However, the target cells of paclitaxel and alloferon-1 are tumor cells and NK cells, respectively, and releasing the two drugs step by step through a nanoparticle is difficult. In addition, PTX is a hydrophobic small molecule, but alloferon-1 is a hydrophilic polypeptide. Using nanoparticles to coload two drugs with distinct physicochemical properties is another problem. Thus, this work

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explored the tumor microenvironment responsiveness of nanosystems.

The matrix in the tumor microenvironment is mainly composed of structural proteins and

glycosaminoglycans, and the main component of the latter is heparan sulfate proteoglycans

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(HSPGs) (14). In the tumor microenvironment, HSPGs are involved with many biological factors

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(such as b-FGF, VEGF, and TGF-β) that are highly strategic in the battle between the body and the

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tumor. However, studies have shown that malignant, highly metastatic tumors express high levels of heparanase-1, especially in melanoma (15). Heparanase-1 in the tumor microenvironment

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usually degrades HSPGs, enabling the massive release of these biological factors and the subsequent activation of signaling pathways leading to neovascularization,

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epithelial-mesenchymal transition (EMT), and tumor metastasis. In addition, the new voids in the matrix also contribute to tumor cell invasion and metastasis (15). Heparanase-1 can degrade

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heparan, but it is not known for the degradation of unfractionated heparin. Ongoing research has shown that heparanase-1 can also degrade heparin, indicating that a new carrier material with enzyme responsiveness has been discovered (16). The first method of applying heparin to nanoparticles is to connect heparin molecules through disulfide bonds to prepare a heparin nanogel (17). However, a different method was chosen for 6

this work, which involved the preparation of a polyelectrolyte complex via electrostatic attraction between negatively charged heparin and positively charged protamine to encapsulate a positively charged alloferon-1 polypeptide. Researchers have reported similar experiments with polyelectrolyte complexes carrying small charged molecules (18). These reports studied the formation mechanism and internal laws of electrostatic adsorption on a microscopic scale using real-time dynamic swelling spectra and isothermal titration calorimetry (19). By optimizing the

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preparation process, a polyelectrolyte complex can be prepared with a controlled particle size and a negative surface charge (20). DOTAP, a positively charged phospholipid molecule commonly

used to prepare cationic liposomes, can be used to entrap hydrophobic PTX by forming micelles

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(21). By optimizing the preparation process, a negatively charged electrolyte complex with a

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suitable particle size completely covers the positively charged DOTAP micelles to ultimately form

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a negatively charged nanoparticle. Once the codelivered PTX and alloferon-1 nanoparticles enter the tumor microenvironment, heparanase-1 rapidly recognizes the outer heparin molecule and

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degrades it, eventually releasing alloferon-1 to activate NK cells. This also causes the positively charged DOTAP core to be exposed by the tumor cells. Using this method, the two problems of

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different target cells and coloading are resolved. Using a thorough analysis consisting of X-ray photoelectron spectroscopy, radioactive labeling of

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external sites, Fourier transform infrared spectroscopy, fluorescence correlation spectroscopy single-molecule polarization resolution spectroscopy, and a novel three-dimensional orientation technique, previous studies found that electrolyte-induced ion diffusion is an important factor that promotes the release of charged drug molecules from polyelectrolyte complexes (22). The synergistic effect of ion diffusion and heparanase-1 is the mechanism that promotes the 7

degradation of nanoparticles and the release of alloferon-1. In summary, the nanoparticle has four advantages. 1) Since there is no chemical synthesis in the nanoparticle preparation process, almost no organic solvent is used and the preparation process is uncomplicated. Nanoparticle size uniformity is good, so the nanoparticles are druggable and can be industrially produced and used for clinical purposes. 2) The reduction in particle size and the ζ-potential inversion of the nanoparticles during the depolymerization of the polyelectrolyte

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complex facilitates the delivery of more nanoparticles to greater tumor depth as well as greater nanoparticle uptake by tumor cells with highly negative charges on the surface (23). 3) The nanoparticles are very intelligently designed, and their procedural release allows PTX and

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alloferon-1 to be released step by step to act on different target cells. 4) Highly biocompatible and

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degradable carrier materials allow nanoparticles to enter clinical trials. DOTAP, a commonly used

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cationic lipid nanomaterial with good biocompatibility, is commonly used by research groups specializing in liposomes (21). Heparin is a biomaterial with significant biocompatibility and high

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degradability (24). Protamine and heparin can also neutralize each other during the formation of polyelectrolyte complexes, which prevents a large dose of protamine and heparin from being

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released in the blood circulation (25). In this experiment, only the outermost layer of heparin in the nanoparticle was exposed to the circulatory system, which significantly reduced the material’s

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side effects.

RESULTS AND DISCUSSION Characterization of PTX-DOTAP@alloferon-1-heparin/protamine A brief flow chart for the preparation of PTX-DOTAP@alloferon-1-heparin/protamine is summarized in Figure 1a. Nanoparticles were prepared by replacing PTX and Alloferon-1 with 8

Coumarin 6 and Rhodamine B, respectively. Fluorescence colocalization experiments showed that Coumarin 6-DOTAP and Rhodamine B-heparin/protamine are located on the same nanoparticle (Figure S1). A concentration of 0.3 mg/ml was used to prepare alloferon-1-heparin/protamine with a small particle size (Figure S2) (20). To obtain a protamine/heparin mass ratio when heparin is saturated, a gradient protamine/heparin mass ratio was designed. There was a slight excess of heparin when the protamine/heparin mass ratio was 0.125 according to the ζ-potential (Figure

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S3a). The excess heparin was dialyzed out in a dialysis bag with a molecular weight of 50 KD. After concentration, the heparin content in the outer solution of the dialysis bag was detected using azure A-heparin colorimetry, and the critical mass ratio of the protamine/heparin was

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calculated to be 13.82:80. The electron micrograph of the nanoparticles showed a round shape and

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uniform particle size (Figure S3b). The particle size was 26.19 ± 1.631 nm (PDI: 0.146 ± 0.018)

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(Figure S3c). The ζ-potential was -42.4 ± 0.246 -mV (Figure S3d). Through a series of single factor investigations and orthogonal design, PTX-DOTAP showed

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relatively good drug loading (7.67% ± 0.01) and encapsulation efficiency (99.70% ± 0.15) when the ratio of drug to lipid was 1:12, the lipid concentration was 1.5 mg/ml, the hydration

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temperature was 35°C, and the ultrasonic time was 50 s. The particle size was 52.70 ± 1.491 nm (PDI: 0.163 ± 0.012) and the ζ-potential was 44.5 ± 0.872mV (Figure S3e and S3f). The electron

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micrograph of PTX-DOTAP showed a round shape and uniform particle size (Figure S3g). Similarly, to obtain the PTX-DOTAP@alloferon-1-heparin/protamine mass ratio, a gradient (heparin/protamine)/DOTAP mass ratio was designed. There was a slight excess of heparin/protamine when the (heparin/protamine)/DOTAP mass ratio was 6 according to the ζ-potential (Figure 1c). The excess heparin/protamine was dialyzed out in a dialysis bag with a 9

molecular weight of 1000 KD. After concentration, the heparin/protamine content in the outer solution of the dialysis bag was detected using azure A-heparin colorimetry, and the critical mass ratio of (heparin/protamine)/DOTAP was calculated to be 4.85. The electron micrograph of the nanoparticles showed a uniform shape and particle size, with no extra heparin/protamine in the background (Figure 1e). The particle size was 106.1 ± 1.113 nm (PDI: 0.147 ± 0.005) (Figure 1d). The ζ-potential was -45.1 ± 0.455 mV (Figure 1f). This property allows the nanoparticles to

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be stable in the blood (Figure S4). The particle size of the nanoparticles according to the electron

micrograph was slightly smaller than that measured by the Malvern particle size analyzer because the latter has a more hydrated layer than the former (21). The relationship between the efficiency

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of nanoparticles targeting tumors and the particle size of the nanoparticles was studied. When the

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particle size was approximately 100 nm, the enrichment of nanoparticles at tumor sites was better

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(26-28).

In addition, nanoparticles were prepared when (heparin/protamine)/DOTAP was lower than 4.85.

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The electron micrographs showed that the nanoparticle size was not uniform and much larger than 100 nm because the nanoparticle contained several DOTAP cores (Figure S5a and S5c). In

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addition, there were many heparin/protamine balls in the background of the electron micrograph when the heparin/protamine balls were not removed using the 1000 KD dialysis bag (Figure S5a

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and S5c).

In vitro release behaviors of PTX-DOTAP@alloferon-1-heparin/protamine Immunofluorescence staining of heparanase-1 showed a large amount of heparanase-1 in the tumor stroma of mice and a small amount of heparanase-1 in the intercellular substance of the liver compared with the normal skin group (Figure 2a). Heparinase can be used to degrade heparin 10

in vitro to mimic heparanase-1 degradation of heparin in vivo (29). In this study, the release of PTX-DOTAP@alloferon-1-heparin/protamine nanoparticles was performed in vitro. A pH of 6.5 was used to simulate the pH in the tumor microenvironment. To mimic the tumor microenvironment, 0.01 nM Tris-HCl was used to mimic the electrolyte environment and 20 nM Ca2+ was used to mimic the Ca2+ concentration in the tumor microenvironment and enhance heparanase-1 activity. In the result (Figure 2b), the blue curve represents the release of PTX, and

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the green curve represents the release of alloferon-1. The released amounts of free alloferon-1 and free PTX within 6 h were 85.22 ± 0.82% and 90.01 ± 1.41%, respectively, and afterward the

concentration in the outer solution of the dialysis bag was almost unchanged, indicating that the

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two groups quickly passed through the 35 KD dialysis bag to the external solution. The

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PTX-DOTAP@alloferon-1-heparin/protamine group supplemented with 50 µl of 0.0833 IU/ml

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heparinase released alloferon-1 from 0 h, and its release rate was significantly reduced at 10 h. The PTX was quickly released from 2 h because the PTX in the core of the nanoparticle was

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protected by the alloferon-1-heparin/protamine layer and was not suddenly released. The release rate was significantly reduced at 16 h. Finally, the released amounts of PTX and alloferon-1 were

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83.82 ± 1.12% and 81.67 ± 0.76%, respectively. In the PTX-DOTAP@alloferon-1-heparin/protamine group untreated with enzyme, only 12.77 ± 1.58%

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PTX and 8.43 ± 1.79% alloferon-1 were released throughout the release experiment, indicating that PTX-DOTAP@alloferon-1-heparin/protamine nanoparticles can provide excellent protection for internal drugs without enzymatic degradation. Observation of phenomena during release and related determination During the release process, the appearance phenomenon and electron micrographs of each group 11

were recorded (Figure 2c). At the beginning, the solution showed a pale blue opalescence due to the presence of nanoparticles. The group without enzymes and electrolytes did not change significantly throughout the process, which indicates stability in pure water. At 5 min, the enzyme group and enzyme + electrolyte group quickly became cloudy, and the solution contained many insoluble white floes. The electron micrographs also showed that a large number of nanoparticles are clustered together. This was because the alloferon-1-heparin/protamine, the outermost layer of

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the PTX-DOTAP@alloferon-1-heparin/protamine nanoparticles, was enzymatically hydrolyzed,

and the heparin was gradually released, exposing a portion of the positively charged protamine. At this time, the nanoparticles with uneven surface charges were combined by electrostatic attraction

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to aggregate into a larger particle size. We believe that the increase in the particle size of the

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nanoparticles in a short period of time prevents them from returning to the blood circulation

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through the pores in the blood vessels at the tumor, thereby contributing to the improved accumulation capacity of the nanoparticles at the tumor site, as demonstrated in previous reports

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(30-32). At 6 h, the turbidity of the enzyme group and the enzyme + electrolyte group drastically decreased, and the white floes in the solution significantly decreased. The electron micrograph at

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this time also showed a nanoparticle aggregate with a smaller particle size than the solution at 5 min. At 6 h, the color of these nanoparticle aggregates was also more transparent and whiter than

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at 5 min in the electron micrograph. This phenomenon indicated that the alloferon-1-heparin/protamine layer of the PTX-DOTAP@alloferon-1-heparin/protamine formulation was significantly degraded by the heparinase and detached by ion diffusion (22, 33). In addition, at 6 h, the electrolyte group became very turbid, and the solution contained a large number of white floes. This was due to the ion diffusion caused by the electrolytes, such that the 12

alloferon-1-heparin/protamine layer of the PTX-DOTAP@alloferon-1-heparin/protamine was also slowly dissociated, causing the surface-discharged nanoparticles to gather together (34). However, without the participation of heparinase, the progress of this physical phenomenon was significantly reduced. At 12 h, the enzyme + electrolyte group became very clear, and the white floes in the solution almost completely disappeared. The electron micrographs also showed that the solution contained only a few independent nanoparticles with particle sizes of approximately

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50 nm. This was the result of the combination of heparinase and ion diffusion, causing the alloferon-1-heparin/protamine layer of the PTX-DOTAP@alloferon-1-heparin/protamine

formulation to almost completely detach, exposing the internal DOTAP core. Moreover, after

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measurement, the particle size and potential of the nanoparticles in the solution were 59.30 ±

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0.783 nm (PDI: 0.234 ± 0.032) and 25.4 ± 0.257 mV, respectively (Figure 2d). Since the

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alloferon-1-heparin/protamine layer of the PTX-DOTAP@alloferon-1-heparin/protamine formulation did not completely degrade, the positive charge of the exposed DOTAP core was

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lower than the positive charge of the DOTAP core during preparation. This undoubtedly proves that enzymatic degradation led to the diffusion of protamine and the weakening of the electrostatic

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attraction between the protamine and the heparin, which contributed to ion diffusion of the electrolyte in the buffer. In turn, ion diffusion promoted further enzymatic degradation of the

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alloferon-1-heparin/protamine layer. This resulted in a positive feedback cycle that promoted the degradation of all of the alloferon-1-heparin/protamine layers by enzymatic degradation and exposed the positively charged DOTAP core. According to the released amount of PTX and alloferon-1 measured by the release experiment, the ion diffusion of the electrolyte alone could not promote the depolymerization of PTX-DOTAP@alloferon-1-heparin/protamine. Only the 13

synergistic effect of the heparinase and ion diffusion caused the formulation to almost completely dissociate, reducing particle size and charge turnover of the nanoparticles. Therefore, when the nanoparticles reach the tumor site, the particle size reduction process will change the particle size of the nanoparticles from approximately 100 nm to 50 nm and increase the number of nanoparticles entering the tumor (23). In addition, due to the high expression of glycoproteins on the surface of the tumor cells, the heparanase-1 degrades the outer layer to promote ion diffusion

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to expose the most positively charged DOTAP core. The positively charged DOTAP is easily

adsorbed and ingested by tumor cells, which enhances the paclitaxel uptake by the tumor cells and improves the efficacy of chemotherapy.

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This work also characterized changes in conductivity (Figure 2e). First, the conductivity of pure

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water was 1.580 us/cm, the conductivity of Tris-HCl buffer was 4160 us/cm, and the conductivity

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of PTX-DOTAP@alloferon-1-heparin/protamine was 303 us/cm. After the addition of the heparinase, the conductivity of the PTX-DOTAP@alloferon-1-heparin/protamine was 304.2 us/cm.

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Although the conductivity of the electrolyte solution was much greater than the conductivity change caused by enzymatic degradation the conductivity of the heparinase group increased first

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within 1 h and then was affected by the buffer medium. This proves that after the nanoparticles were hydrolyzed by the enzyme, the nanoparticles were depolymerized and the electrolytes in the

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solution increased.

In this study, the nanoparticles were prepared using sodium hyaluronate instead of heparin sodium, and changes in the conductivity of the enzyme-added solution were detected. This involved a group of isotype control experiments where pure water was used as a buffer (Figure 2f). The conductivity data demonstrated that the initial conductivity of the 14

PTX-DOTAP@alloferon-1-heparin/protamine and PTX-DOTAP@alloferon-1-hyaluronate/protamine was 34.6 ± 1.1 (us/cm) and 36.6 ± 0.9 (us/cm), respectively, but within 15 min of the addition of heparinase and hyaluronidase, both solutions became very turbid and the conductivity in the solution was 447.0 ± 1.9 (us/cm) and 109.1 ± 1.7 (us/cm), respectively. The two groups of solutions were then dialyzed (35 KD) for 1 h, and the conductivity in the solution was 52.4 ± 1.3 (us/cm) and 68.6 ± 0.8 (us/cm), respectively. This

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proved that the electrolyte complex was destroyed during the enzymatic degradation process and

released a significant number of electrolytes, which passed through the 35 KD dialysis bag. Hence, the experiment in this study was redesigned. At 24 h, the two groups were centrifuged and the

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measured conductivity of the supernatants was 472.0 ± 1.2 (us/cm) and 106.5 ± 1.5 (us/cm),

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respectively. After the suspension was resuspended, the solution remained turbid and the measured

aforementioned theory.

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conductivity was 15.7 ± 1.7 (us/cm) and 5.07 ± 0.5 (us/cm), respectively. This again validates the

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Evaluation of PTX-DOTAP@alloferon-1-heparin/protamine uptake by B16F10 cells Coumarin 6 is a fat-soluble fluorescent dye commonly used in experiments to evaluate cellular

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uptake. The following conclusions could be drawn from fluorescence microscopy and flow cytometry semiquantitative fluorescence intensity (Figure 3a). Coumarin 6 enters the cell through

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passive diffusion and its cell uptake efficiency is slightly lower than that of nanoparticle-mediated cell endocytosis, so the free C6 group has the lowest fluorescence intensity. To simulate the cell uptake of positively charged DOTAP cores exposed by enzymatic degradation of PTX-DOTAP@alloferon-1-heparin/protamine in the tumor microenvironment, the cell uptake of C6-DOTAP group was studied in vitro. The fluorescence intensity of this group was the strongest, 15

indicating that the positive charge increased the cell uptake efficiency of the nanoparticles. The two coumarin 6-HEP NP and heparin preincubation + coumarin 6-HEP NP experiments demonstrated that the uptake of the nanoparticles by the cells was much higher than that of free coumarin 6. Preincubating the cells with heparin did not affect the uptake of coumarin 6-HEP NP by the cells, indicating that the heparin molecule did not target tumor cells. Finally, the uptake of coumarin 6-CS NP and coumarin 6-HA NP was assessed. Compared with the coumarin 6-HEP NP,

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the results showed that hyaluronic acid and chondroitin sulfate have a certain targeting effect on tumor cells and enhanced tumor cell uptake of the nanoparticles. The results of this experiment

provide a theoretical basis for a subsequent group of in vivo imaging experiments. In addition, this

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work also semiquantified the uptake of these groups, and the results were consistent with those

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observed by fluorescence microscopy (Figure 3b and 3c).

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Cytotoxicity assay of PTX-DOTAP@alloferon-1-heparin/protamine in vitro The cytotoxicity of different formulations was determined using the CCK-8 method. The toxicity

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of the blank NP showed that the nanocarriers composed of heparin, protamine, and DOTAP were almost nontoxic to tumor cells, even at a carrier concentration of 14000 ug/ml, and at 24 h and 48

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h there was no significant difference in the experimental results between the two (Figure 4a). In addition, the toxicity test results of the free drug or nanoparticle groups showed that the cell

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viability value at 48 h was less than at 24 h, indicating that the toxicity of the preparation increased with time (Figure 4b and 4c). First, the PTX/alloferon-1 NP was much more toxic to cells than the free combo, which proved that the uptake of the nanoparticles by the cells was much stronger than the uptake of the free drugs. Second, the experimental results of the free alloferon-1 group showed that alloferon-1 was almost nontoxic to the cells, and even if the concentration 16

reached a maximum, the cell viability was only slightly lower than 90%. The toxicity of PTX was a key factor in the overall cytotoxicity results. According to the experimental results, the free IC50 of the free PTX, free combo, PTX NP, and PTX/alloferon-1 NP was 57.22 ug/ml, 52.49 ug/ml, 14.42 ug/ml, and 13.16 ug/ml, respectively. The apoptosis rate is a common indicator to evaluate the effects of the formulation on cytotoxicity. A 6 ug/ml PTX concentration with low toxicity was used in the apoptosis experiment. As shown in

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Figure 4d and 4e, the cell survival rate without PTX was 98.42%, and the cell survival rate of the blank NP group was 96.31%, indicating that the blank NP had almost no cytotoxicity. Moreover, the cytotoxicity of the alloferon-1 was also relatively low, again confirming that the polypeptide

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was almost nontoxic to the B16F10 cells. The cell survival rate of the nanoparticle group was

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lower than that of the free drug group, which also confirmed that the uptake of nanoparticles by

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the cells was stronger than that of the free drug group. In general, PTX occupies a key position in the process of killing cells using drugs, and the apoptosis results were consistent with the results

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of the CCK8 cytotoxicity experiments.

Cell cycle experiments are a powerful weapon to study the mechanism of action of drugs. The

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results of the effects of each drug group on the B16F10 cell cycle are shown in Figure 4f and 4g. The results indicated that PTX arrested the cell proliferation cycle in the G2/M phase, especially

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PTX/alloferon-1 NP, which confirmed that the nanoparticle form increased the uptake of the drug by the cells. In addition, comparing the alloferon-1 NP and control groups indicated that alloferon-1 slightly arrested cells in the S phase, but drug efficacy was not strong. Tumor xenograft mouse model and assay of biodistribution in mice To observe the real-time distribution of drugs and nanoparticles in the body, tumors, and major 17

organs, we used an in vivo imaging experiment to analyze the relevant results. The excitation wavelength of DiR fluorescent dye exceeded 650 nm; its fluorescence can effectively penetrate muscle tissue and skin tissue and was sensitively detected using live mouse imaging. As shown in Figure 5a, the free-DiR, DiR-HEP/DOTAP, DiR-DOTAP, and DiR-HA/DOTAP groups had different dynamic distributions in the mice. The free-DiR results showed that the DIR without nanoparticle entrapment had a systemic distribution, and compared with the other three groups, it

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crossed the blood-brain barrier and a large amount accumulated in the brain of the mice at 24 h.

The DiR-DOTAP results demonstrated that it did not circulate for very long because the positive charge on its surface caused DiR-DOTAP to be rapidly recognized by the reticuloendothelial

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system and phagocytic cells and consequently transported to the liver for metabolism. The

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DiR-HEP/DOTAP and DiR-HA/DOTAP groups had circulated for much longer and showed

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accumulation at the tumor at 24 h. The results also showed that the free-DiR, DiR-DOTAP and DiR-HA/DOTAP showed more major organ accumulation, while DiR-HEP/DOTAP showed less

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liver accumulation (Figure 5b). The major reason for this is because the tumor has high expression of heparanase-1, which could degrade the nanoparticles resulting in high accumulation of

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nanoparticles and more tumor cell uptake at the tumor site. In contrast, the accumulation of nanoparticles in the liver would be reduced. In addition, according to the "Guidelines for the Use

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of Spinal Blocks in Patients Receiving Anticoagulant Therapy" published by the National Association of Local Anesthesiology and Pain Medicine in May 2003 (24), the results of heparanase-1 expression based on BioGPS (35) indicated that the liver has a certain degree of heparanase-1 expression (Figure 3a). The minor reason is that heparanase-1 in the liver could degrade DiR-HEP/DOTAP, which would accelerate the metabolic processing of nanoparticles. 18

Finally, the liver fluorescence intensity of the DiR-HEP/DOTAP group was lower than that of the DiR-HA/DOTAP group (Figure 5c). The DiR fluorescence intensity of the liver in the DiR-DOTAP group was high because the accumulation of DiR-DOTAP in the liver was much greater than that of the DiR-HEP/DOTAP group. The cell targeting effect of coumarin 6-HA/DOTAP was much better than the coumarin 6-HEP/DOTAP group based on previous cellular uptake experiments. However, this study found that the fluorescence intensity of

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DiR-HEP/DOTAP at the tumor site was stronger than that of the DiR-HA/DOTAP group. This phenomenon indicates that heparanase-1 degrades DiR-HEP/DOTAP in vivo. Specifically, the

initial aggregation of nanoparticles increases retention, the particle size decreases and the charge

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turnover increase the uptake by tumor cells. These conclusions are consistent with the

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semiquantitative results (Figure 5c).

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Antitumor study in vivo

To further evaluate the therapeutic effect of each formulation group on tumors in mice, PBS, blank

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NP, free PTX, free alloferon-1, free combo, PTX NP, alloferon-1 NP, and PTX/alloferon-1 NP groups were designed and tested (n = 8 per group). When the subcutaneous tumor volume of the

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mice reached 100 mm3, intravenous administration was initiated. The mice were administered the drugs once every three days and the relevant data were recorded. The results showed that

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PTX/alloferon-1 NP had the best inhibitory effect on tumors, suggesting that the combination of PTX and alloferon-1 was stronger than the two drugs alone (Figure 6a and 6b). It was obvious that the therapeutic effect of the nanoparticle group was much greater than that of the free drug group, indicating that the passive targeting of the nanoparticles enhanced the efficacy of the drug. Unlike the results of the cytotoxicity experiments, the alloferon-1 NP group showed some tumor 19

inhibition, suggesting that alloferon-1 inhibits tumor growth in an indirect way, activating NK cells and reversing immune system inhibition mediated by the tumor microenvironment. The blank NP also showed some tumor suppressing activity. There are two reasons for this observation. On one hand, heparin in the outer layer of the blank NP could bind to platelets, thereby preventing the combination of tumor cells and platelets from forming circulating tumor cells, which inhibits tumor metastasis and further deterioration of the tumor to some extent (36). On the other hand, the

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fact that blank NP can be degraded by heparanase-1 also showed that the blank NP can act as a substrate for heparanase-1, inhibiting the enzymatic activity of heparanase-1 to HPSGs and reducing the release of VEGF and b-FGF angiogenesis factors, thereby inhibiting tumor

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metastasis and further deterioration of the tumor to some extent. Changes in the body weight of

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the mice were recorded (Figure 6c). The results showed that the free PTX and free combo groups

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seriously affected the normal growth of the mice in the middle of drug administration, while the other groups showed no significant effect on the normal growth of the mice. This shows that the

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encapsulation function of the nanoparticles will significantly reduce the damage by the chemotherapeutic drug PTX to the body. The study of the survival time (Figure 6d) demonstrated

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that PTX/alloferon-1 NP had the best therapeutic effect and significantly prolonged the survival of the mice, which is consistent with the results of the tumor inhibition rate. At the end of the

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administration process, the mice in the free PTX and free combo groups had low body temperatures and very low vitality compared with the mice in the PTX/alloferon-1 NP group. Similar to the normal mice, the general body temperatures of the mice in the PTX/alloferon-1 NP group were normal and their vitality was very strong. Immunohistochemistry and immunofluorescence staining on tumor sections from the mice were 20

also performed. Ki67 immunohistochemistry showed the fluorescence intensity of the proliferating cells while TUNEL staining indicated the fluorescence intensity of the apoptotic cells. These two opposing experimental methods demonstrated tumor cell apoptosis in vivo in each of the preparation groups. The PTX/alloferon-1 NP group had the strongest pro-apoptotic ability in the tumor cells in vivo and was significantly different than the other groups (Figure 6e and 6f). This result is consistent with the conclusion of the previous tumor inhibition rate experiment.

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Immune-related research in vivo

Studies have reported that alloferon-1 does not directly kill tumor cells, but only activates NK

cells that are inhibited by tumor cells, thereby indirectly killing tumor cells through the immune

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system (10, 37). The previous B16F10 cytotoxicity test could not test the cancer treatment effect

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of alloferon-1. Animal experiments are the most reliable tests to prove the efficacy of a certain

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drug, so some correlated evaluations on the immune effect of alloferon-1 in vivo were conducted in this study.

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IFN-γ and TNF-α are biological factors that can kill a large amount of tumor cells and activate other lymphocytes (such as T and B lymphocytes). When NK cells are activated, the amount of

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IFN-γ and TNF-α released from the cells is significantly increased (38). Therefore, an ELISA kit was used in this work to study the levels of IFN-γ and TNF-α in the peripheral blood of the mice.

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The results indicated that the formulation groups containing alloferon-1 increased the concentration of IFN-γ and TNF-α in the blood. The PTX/alloferon-1 NP group had the best efficacy (Figure 7a and 7b). Consistent with the aforementioned experimental results, the tumor sections underwent immunofluorescence staining using anti-IFN-γ mAb and anti-TNF-α mAb double staining (Figure 7c). The results inferred that PTX/alloferon-1 NP promoted the release of 21

IFN-γ and TNF-α by NK cells, and the fluorescence intensity of all of the preparation groups containing alloferon-1 were also strong, which is consistent with the aforementioned results. Subsequently, anti-NKG2D mAb and anti-CD94 mAb were applied to the immunofluorescence staining of the tumor sections. NKG2D is an activating receptor that is highly expressed when NK cells are activated, and CD94 is an inhibitory receptor that is highly expressed when NK cells are inhibited (38). As the results demonstrated (Figure 7d and 7e), PTX/alloferon-1 NP showed the

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strongest fluorescence intensity with NKG2D and CD94, and the fluorescence intensity of all of the preparation groups containing alloferon-1 were also strong. It may be speculated that

alloferon-1 activated NK cells causing release of a considerable amount of IFN-γ and TNF-α in

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vivo, which is consistent with the aforementioned results (Figure 7f and 7g).

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To investigate the overall immune system recovery in the tumor microenvironment, the tumor

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sections also underwent immunofluorescence staining using anti-CD8 mAb and anti-granzyme B mAb double staining. Testing the expression levels of CD8 and granzyme B provides a more

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detailed understanding of the recovery of the immune system following tumor suppression (38). The results (Figure 7h) showed that the fluorescence intensity of CD8 and granzyme B in the

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PTX/alloferon-1 group was the strongest and that the fluorescence intensity of all of the preparation groups containing alloferon-1 was also strong, indicating that alloferon-1 activated

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lymphocytes (such as T and B cells) by activating NK cells and reversed the immune inhibition mediated by tumor cells. This is consistent with the previous immunoassay results. Toxicity study of PTX-DOTAP@alloferon-1-heparin/protamine in major organs In general, hematoxylin and eosin staining of vital organs is an important means to evaluate drug toxicity. One goal of this study was to demonstrate that the toxicity of chemotherapeutic drugs 22

encapsulated by nanoparticles was significantly reduced compared to that of free chemotherapeutic drugs and that the damage to organs would be negligible. In Figure 8, as demonstrated by the red arrow, small focal inflammation and hepatocyte coagulation were observed in the liver and spleen and lung congestion was also present, indicating a slight degree of damage and necrosis of the liver, spleen, and lungs. In addition, renal edema was relatively severe, and edema, hemorrhage, and luminal occlusion were observed in the proximal tubules, suggesting

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severe systemic toxicity by the free drugs. This indicates that the damage to the organs caused by the free PTX was far stronger than that of the other preparation groups, which further proves that to reduce the damage of chemotherapeutic drugs to the body, nanotechnology is an important

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method that cannot be ignored. In terms of serum testing, the concentrations of aspartate

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aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) in the

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peripheral blood were measured (Table S1). The results indicated that the influence of each formulation group on liver and kidney function were largely insignificant. Only two groups, the

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free PTX and free combo, had slightly enhanced plasma concentrations of AST, ALT, and BUN. In the PTX/alloferon-1 NP group, since the size of the nanoparticles was approximately 100 nm and

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the surface had a strong negative charge, little toxicity organ toxicity was observed. CONCLUSIONS

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In this study, a PTX-DOTAP@alloferon-1-heparin/protamine nanosystem was successfully designed. This work demonstrated that it could be an excellent template for the treatment of highly metastatic tumors with high expression of heparanase-1. The particle size of approximately 100 nm and the negative charge protection by heparin engenders high stability in the circulatory system and high enrichment of the nanoparticles at the tumor site. Subsequently, heparanase-1 in 23

the tumor microenvironment rapidly degrades heparin in the outer layer of the nanoparticles. The ion diffusion caused by the electrolytes in the interstitial fluid dominates the nanoparticle depolymerization process. During this process, the nanoparticles will release alloferon-1 to activate NK cells to reverse the immune system inhibition mediated by tumor cells. Under the dual action of enzymatic hydrolysis and ion diffusion, the internally positively charged DOTAP core is exposed. Nanoparticles with a reduced particle size and positive charge could penetrate into the

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interior of the tumor and are more easily endocytosed by highly negatively charged tumor cells, thereby improving the therapeutic effect of the chemotherapeutic drug. Moreover, in the

nanoparticle design process, the toxicity of the carrier material is significantly reduced, which

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provides a favorable basis for clinical research. Cell and animal experiments have shown that a

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combination of chemotherapy and immunotherapy is an important component of future cancer

EXPERIMENTAL SECTION

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Materials

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treatment.

Paclitaxel, alloferon-1, heparin sodium, and protamine sulfate were purchased from Dalian Meilun

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Biotechnology Co. Ltd. (Dalian, China). DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) was purchased form Corden Pharma (Liestal, Switzerland).

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Cell lines

A B16-F10 cell line (mouse melanoma cells) was purchased from Procell Life Science & Technology (Wuhan, China) and cultured in H-Dulbecco modified Eagle medium supplemented 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated at 37°C in the presence of 5% CO2. 24

Preparation of PTX-DOTAP A total of 0.550 ml of 1 mg/ml PTX (dissolved in chloroform) and 6.616 ml of 1 mg/ml DOTAP (dissolved in chloroform) were uniformly mixed and then rotary evaporated at room temperature for 15 min. Subsequently, 4.411 ml of water was slowly added to a flask under ultrasound at 30°C and ultrasonicated for 50 s. Preparation of alloferon-1-heparin/protamine polyelectrolyte complex

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After mixing 15.33 ml of 0.3 mg/ml protamine sulfate and 0.42 ml of 0.3 mg/ml alloferon-1, the mixture was slowly added dropwise to 91.215 ml of 0.3 mg/ml heparin sodium solution at room temperature and stirred for 30 min (600 rpm).

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Preparation of PTX-DOTAP@alloferon-1-heparin/protamine

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A total of 4.411 ml PTX-DOTAP was slowly added dropwise to 106.97 ml of

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alloferon-1-heparin/protamine polyelectrolyte complex at room temperature and stirred for 10 min (200 rpm). It was then dialyzed for 10 min in a 1000 KD dialysis bag to remove excess

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alloferon-1-heparin/protamine.

Characterization of PTX-DOTAP@alloferon-1-heparin/protamine

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The particle size, size distributions, and ζ-potentials of PTX-DOTAP, alloferon-1-heparin/protamine and PTX-DOTAP@alloferon-1-heparin/protamine were measured

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using a Zetasizer Nano ZS90 instrument (Malvern, Westborough, MA, USA) by the dynamic light-scattering method. To observe the morphology, the solution containing nanoparticles was dropped onto a copper mesh and stained with 2% (w/v) phosphotungstic acid hydrate. After 30 min of drying, the sample was observed under a transmission electron microscope (JEM 1200EX, JEOL, Tokyo, Japan) with an accelerating voltage of 80 kV. The measurements were performed in 25

triplicate in independent experiments. In vitro release behaviors of PTX-DOTAP@alloferon-1-heparin/protamine The dynamic dialysis method was used to assess the release behavior of PTX and alloferon-1 in the presence or absence of heparinase in vitro. In brief, aliquots of samples were placed into a dialysis bag (with a cutoff molecular weight of 35 KD) and dialyzed against 0.01 nM Tris-HCl containing 0.5% Tween-80 and 20 nM Ca2+ (pH = 6.5), which can simulate tumor release

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behavior. Next, the dialysis bag was stirred at 100 rpm at 37°C and 1 mL of dialysate was

removed from the sample at predetermined intervals. To maintain a constant volume, an equal

volume of fresh medium with corresponding pH was added. The concentrations of the released

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PTX and alloferon-1 were determined by HPLC using the LC 1200 HPLC system (Agilent, Santa

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Clara, CA, USA). The chromatographic conditions for PTX were as follows: λ max = 227 nm, mobile phase, and acetonitrile: water = 70:30 (v/v). The chromatographic conditions for

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alloferon-1 were as follows: λ max = 232 nm, mobile phase, and acetonitrile: water = 10:90 (v/v).

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The analyses were measured in triplicate.

Observation of phenomena during release and related determination

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We observed the appearance phenomenon in the dialysis bags of each group throughout the release process, including the turbidity, color, and Tyndall phenomenon, and detected the particle

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size, ζ-potential (Zetasizer Nano ZS90 instrument), and morphology (using a transmission electron microscope system) of the nanoparticles in the heparanase-1 preparation group at the corresponding time points. PTX-DOTAP@alloferon-1-hyaluronic acid/protamine was prepared via the same preparation method by replacing sodium heparin with sodium hyaluronate. Heparanase-1 and hyaluronidase 26

were added to PTX-DOTAP@alloferon-1-heparin/protamine and PTX-DOTAP@alloferon-1-hyaluronic acid/protamine, respectively. The conductivity of both at 15 min and after dialysis (35 KD) for 1 h were measured using a conductivity meter (Mettler Toledo, Zurich, Switzerland). After 24 h of enzyme addition, the two groups were centrifuged (3000 rpm), and the conductivities of the supernatant and the redispersed precipitate were measured via a conductivity meter (Mettler Toledo, Zurich, Switzerland).

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Evaluation of PTX-DOTAP@alloferon-1-heparin/protamine uptake by B16F10 cells

Coumarin 6 (C6) as a lipophilic fluorescent probe can be used to analyze cellular drug uptake. The B16-F10 cells were seeded into six-well plates (Corning Inc., New York, NY, USA) at a density of

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5 × 105 cells/well in complete Dulbecco modified Eagle medium containing 10% fetal bovine

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serum and incubated overnight. After washing twice with PBS, the cells were treated with free C6,

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C6-DOTAP, C6-HEP NP (C6-DOTAP@heparin/protamine), heparin preincubation + C6-HEP NP, C6-CS NP (C6-DOTAP@chondroitin sulfate/protamine), and C6-HA NP

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(C6-DOTAP@hyaluronic acid/protamine) for 4 h at 37°C. The final concentration of C6 was 1 μg/mL. The cells were then washed twice with PBS and fixed with 4% (w/v) paraformaldehyde.

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For visualization, the cells were incubated with DAPI (10 μg/mL) for 15 min and rewashed 3 times with PBS. The cellular uptake images of different formulations of the B16-F10 cells were

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visualized with a fluorescence microscope (IX 70-142, Olympus Corp., Tokyo, Japan). To further quantify the uptake efficiency of the cells, they were detached using 200 μL of trypsin-EDTA solution and centrifuged at 1000 rpm for 5 min, transferred into flow tubes (Falcon, Corning, New York, NY, USA), resuspended in PBS at 1 × 106 cells/mL, and analyzed by flow cytometry (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA). Each experiment was independently 27

performed in triplicate. All of the analyses were processed with CytExpert 3.0 software. Cytotoxicity assay of PTX-DOTAP@alloferon-1-heparin/protamine in vitro The in vitro cytotoxicity of different nanoparticles was measured using a Cell Counting Kit (CCK)-8 according to the manufacturer's protocol (Cell Counting Kit-8, BestBio, Shanghai, China). The B16-F10 cells were seeded in 96-well plates at 37°C at a density of 6000 cells/well in 100 μL complete medium and incubated overnight. The blank NP (DOTAP@heparin/protamine),

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free PTX, free alloferon-1, free combo, PTX NP (PTX-DOTAP@heparin/protamine), alloferon-1 NP (DOTAP@alloferon-1-heparin/protamine), PTX/alloferon-1 NP

(PTX-DOTAP@alloferon-1-heparin/protamine), and control group (with an equal volume of blank

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complete medium added) were then added into each well at the designated concentrations of 0.003,

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0.03, 0.3, 3, 30, and 300 μg/mL for equivalent paclitaxel (the concentration of alloferon-1 was one eighth of paclitaxel) and 0.14, 1.4, 14, 140, and 1400 μg/mL blank nanoparticles and incubated at

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37°C with 5% CO2 for 24 h or 48 h. Before analysis, CCK-8 solution (10 μL/well) was added

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followed by further incubation for 1 h. The maximum absorbance was set at 450 nm, and the optical density (OD) of each well was scanned on a microplate reader (BioTek Synergy H1,

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BioTek Instruments, Inc., Winooski, VT, USA). The relative cell viability (RCV) (%) was calculated as RCV (%) = OD test/OD control × 100%, where OD test and OD control represent

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the OD of the cells treated with the test groups and the control group, respectively. The experiments were repeated six times independently, and the half maximal inhibitory concentration (IC50) of the test groups was calculated using GraphPad Prism 7.0 software. To explore the apoptosis-inducing properties in vitro, the B16-F10 cells were seeded in 12-well plates at a density of 2 × 105 cells/well. After co-culture with different groups of free drugs or 28

nanoparticles, the cells were detached using 100 μL EDTA-free trypsin, washed twice with precooled PBS, and resuspended in 400 μL of binding buffer at a density of 5 × 105 cells/mL. Afterward, the cells were incubated continuously with 5 μL annexin V-FITC for 15 min and 10 μL propidium iodide for 5 min in the dark. The cells were transferred to a flow tube and detected within 30 min using flow cytometry (CytExpert 3.0 software). The B16-F10 cells at 5 × 105 cells/well were added to 6-well plates and treated with different

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groups of free drugs or nanoparticles. After 24 h of incubation, the cells were collected and rinsed with PBS followed by fixation in precooled 70% (v/v) ethanol for 4 h at -20°C. The fixed cells were washed with PBS and harvested with 20 ul RNase at 37°C for 30 min, followed by the

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addition of 400 ul propidium iodide (50 ug/mL) and incubation for another 1 h. The cells were

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then resuspended in PBS, and the propidium iodide intensity and distribution were analyzed using

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flow cytometry (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed and processed with ModFit 3.1 software.

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Tumor xenograft mouse model and assay of biodistribution in mice All of the in vivo mice experiments were approved by the Shandong University Animal Care and

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Use Committee. C57BL/6 mice (6-8 weeks old) were subcutaneously injected with 1 × 106 B16-F10 cells on the right flank. To investigate the drug biodistribution and tumor targeting

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efficacy in vivo, the B16-F10 tumor-bearing mice were randomly grouped (n = 3) when the tumor volume reached 200 mm3, intravenously injected with free DiR, DiR-HEP/DOTAP (DiR-DOTAP@heparin/protamine), DiR-DOTAP, and DiR-HA/DOTAP (DiR-DOTAP@hyaluronic acid/protamine) at 100 μg/kg of the DiR dose. The mice were anesthetized with 4% chloral hydrate at 1 h, 4 h, 12 h, and 24 h post-administration at a dose of 10 29

mL/kg. Real-time images were acquired via the Xenogen IVIS Lumina system (Caliper Life Sciences, Waltham, MA, USA). After 24 h, the mice were sacrificed by cervical dislocation, and the heart, lungs, liver, spleen, kidneys, and tumors were harvested for ex vivo imaging. The images were analyzed in Living Image 4.1 software (Caliper Life Sciences, Waltham, MA, USA) by defining a region of interest around the initial injection site that was then duplicated in all of the images.

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Antitumor study in vivo

To evaluate the antitumor efficacy and safety of the nanoparticles, 1 × 106 B16-F10 cells were

subcutaneously injected into each C57BL/6 mouse. The mice were randomly divided into eight

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groups (n = 8) when the tumor volume reached 100 mm3 and intravenously injected with 100 μL

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of PBS, blank NP (DOTAP@heparin/protamine), free PTX, free alloferon-1, free combo, PTX NP

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(PTX-DOTAP@heparin/protamine), alloferon-1 NP (DOTAP@alloferon-1-heparin/protamine), and PTX/alloferon-1 NP (PTX-DOTAP@alloferon-1-heparin/protamine) at a paclitaxel dose of 10

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mg/kg and an alloferon-1 dose of 1.25 mg/kg. The treatments were performed at time intervals of 3 days. The tumor volume and weight were measured every 3 days to observe the antitumor

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efficacy and toxicity in vivo. The tumor size was measured using a caliper, and the tumor volumes were determined by the formulation V = L × W × W/2 (where L is the longest dimension and W is

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the shortest dimension). At the conclusion of the antitumor study, all of the tumors and vital organs (heart, liver, spleen, lungs, and kidneys) were harvested and fixed with 4% (w/v) paraformaldehyde for further TUNEL, Ki67, and heparanese-1 staining. The survival times of the mice were analyzed following Kaplan-Meier survival fractions using GraphPad Prism 7.0. Immune related research in vivo 30

To explore the NK cells and CD8+ T cell activation in vivo, granzyme B, NKG2D, CD94, IFN-γ, and TNF-α were used to evaluate the alloferon-1 induced antitumor immune response. B16-F10 bearing C57BL/6 mice were intravenously injected with different formulations at the previously mentioned times. Then the mice were sacrificed, the tumor tissues were dissected for immunohistochemistry staining, and the peripheral serum immune factors were tested using the corresponding ELISA kits. Anti-mouse mAbs were used in this study. Anti-granzyme B,

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anti-CD8+, anti-NKG2D, anti-CD94, anti-IFN-γ, anti-TNF-α, IFN-γ ELISA, and TNF-α ELISA

were purchased from Abcam (Cambridge, UK) and HuaBio (Hangzhou, China), respectively. The images were acquired using a laser scanning confocal microscope (LSM 780, Carl Zeiss,

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Oberkochen Germany).

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Toxicity study of PTX-DOTAP@alloferon-1-heparin/protamine on major organs

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To examine the safety of the nanoparticles in vivo, the fixed vital organs (heart, liver, spleen, lungs, and kidneys) were embedded in paraffin, sectioned, and stained with hematoxylin and eosin

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according to the manufacturer's instructions. The stained sections were observed and photographed using a VS120 virtual slide microscope (Olympus Corp., Tokyo, Japan).

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The peripheral serum indicators were tested using an alanine aminotransferase (ALT) ELISA kit, an aspartate aminotransferase (AST) ELISA kit, and a blood urea nitrogen (BUN) ELISA kit.

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Statistical analysis

The data were presented as mean ± standard deviation. One- and two-way analysis of variance was used for multiple comparisons. Bonferroni post-tests were performed when comparing all groups, and the two-tailed t-test was used when comparing two groups. The in vivo tumor treatment studies were repeated in two independent experiments to ensure adequate sample size 31

and reproducibility. All of the statistical analyses were performed using GraphPad Prism and SPSS 19.0 software. Statistical significance was noted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.

ASSOCIATED CONTENT Supplementary data to this article can be found online at

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CONFLICT OF INTEREST STATEMENT The authors declare no competing financial interest. ETHICAL CONDUCT OF RESEARCH

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The authors state their adherence to NIH Guide for the Care and Use of Laboratory Animals.

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STATEMENT

including the Internet.

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ACKNOWLEDGEMENTS

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The authors state that none of the material has been published or is under consideration elsewhere,

This work was supported by the Fundamental Research Funds of Shandong University (No.

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2018JC006), the National Natural Science Foundation of China (No. 21873057), Shandong Provincial Natural Science Foundation of China (No. ZR2019MB041) and the Major Basic

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Research Project of Shandong Natural Science Foundation, P.R.China (No. ZR2018ZC0232).

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Figure legends Figure 1. The preparation process and characterization of PTX-DOTAP@Alloferon-1-Heparin/Protamine. a) The preparation process of PTX-DOTAP@Alloferon-1-Heparin/Protamine. b) During the particle size reduction and charge turnover of PTX-DOTAP@Alloferon-1-Heparin/Protamine, PTX and alloferon-1 are distributed to different target cells. c) Optimal ratio screening of

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PTX-DOTAP@Alloferon-1-Heparin/Protamine. d) The TEM photograph distribution of PTX-DOTAP@Alloferon-1-Heparin/Protamine (n=6). e) The size distribution of

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PTX-DOTAP@Alloferon-1-Heparin/Protamine (n=3).

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PTX-DOTAP@Alloferon-1-Heparin/Protamine (n=3, scale bar is 200μm). f) The ζ-potential of

Figure 2. Heparanase-1 and ion diffusion promote PTX-DOTAP@Alloferon-1-Heparin/Protamine dissociation, appearance phenomenon and conductivity measurements during PTX-DOTAP@Alloferon-1-Heparin/Protamine release. a) The amount of heparanase-1 expressed 38

in B16F10 melanoma, liver and normal skin tissues (n=3, scale bar is 100μm). b) In vitro release profiles of free DTX, free alloferon-1 and PTX-DOTAP@Alloferon-1-Heparin/Protamine in the presence or absence of heparinase at 37 °C. The blue line represents PTX and the green line represents allferon-1. (n= 3, results were shown in mean ± SD). c) In the four buffer environments of none, enzyme, electrolyte and enzyme + electrolyte, PTX-DOTAP@Alloferon-1-Heparin/Protamine exhibited appearance phenomenon, TEM

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photographs and schematic animations at different times (n=3). d) Characterization of the particle size distribution and ζ-potential of the nanoparticles in the enzyme + electrolyte group at 24 h (n=3). e) Conductivity characterization of the four groups of none, enzyme, electrolyte and

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enzyme + electrolyte at different time points (n=3). f) Conductivity of

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PTX-DOTAP@Alloferon-1-Heparin/Protamine and PTX-DOTAP@Alloferon-1-hyaluronic

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acid/Protamine under different treatments (n=3). (results were shown in mean ± SD).

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Figure 3. In vitro cellular uptake experiments of different nanoparticles. a) Cellular uptake

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fluorescence photographs after incubated with free Courmarin-6 (C6), C6-DOTAP, C6-HEP NP (Heparin), heparin preincubation+C6-HEP NP, C6-CS NP (Chondroitin sulfate), C6-HA NP

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(Hyaluronic acid) for 4h at 37°C, cell nucleuses were counterstained with DAPI (10 μg/mL) and the fluorescence signal of DAPI (blue) was merged with C6 (green) of B16-F10 cells (n=6). b)

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and c) Flow cytometry histograms of different nanoparticles (n=3). (scale bar is 100μm)

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Figure 4. In vitro cytotoxicity of different nanoparticles. a) Cell viability of B16-F10 cells after incubated with blank vectors without drug (n=3). b) Cell viability of B16-F10 cells after incubated with free PTX, free Alloferon-1, free Combo, PTX NP (PTX-DOTAP@ Heparin/Protamine),

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Alloferon-1 NP (DOTAP@Alloferon-1-Heparin/Protamine) and PTX/Alloferon-1 NP

(PTX-DOTAP@Alloferon-1-Heparin/Protamine) for 24 h (n=5). c) Cell viability of B16-F10 cells

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after incubated with free PTX, free Alloferon-1, free Combo, PTX NP (PTX-DOTAP@

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Heparin/Protamine), Alloferon-1 NP (DOTAP@Alloferon-1-Heparin/Protamine), PTX/Alloferon-1 NP (PTX-DOTAP@Alloferon-1-Heparin/Protamine) for 48 h (n =5). d) Cell

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apoptosis study of B16-F10 cells after incubation with various free drug and formulations (n=5). e) The apoptotic rate after treatment with various free drug and formulations (n=5). f) Cell cycle

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change study of B16-F10 cells after incubation with various free drug and formulations (n=3). g)

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The cell cycle change rate after treatment with various free drug and formulations (n=3). (results were shown in mean ± S.D., ‘*’ indicates P < 0.05, ‘**’ indicates P < 0.01 and ‘***’ indicates P < 0.001.)

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Figure 5. Real-time monitoring of different formulations after administration. a) In vivo

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fluorescence images of mice bearing B16-F10 melanoma after intravenous injection of free DiR, DiR-HEP/DOTAP (DiR-DOTAP@Heparin/Protamine), DiR-DOTAP and DiR-HA/DOTAP

(DiR-DOTAP@Hyaluronic acid/Protamine) for 1h, 4h, 12h and 24h. The dashed circles indicate

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the tumor foci of mice. b) Ex vivo distribution and c) statistical analysis of hearts (H), livers (Li), spleens (S), lungs (Lu), kidneys (K) and tumors (T). (n= 3, results were shown in mean ± S.D.,

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‘***’ indicates P < 0.001.)

Figure 6. PTX-DOTAP@Alloferon-1-Heparin/Protamine enhance tumor inhibition and delay tumor growth. a) Final tumor volume after treatment of PBS, blank NP (DOTAP@ Heparin/Protamine), free PTX, free Alloferon-1, free Combo, PTX NP (PTX-DOTAP@ Heparin/Protamine), Alloferon-1 NP (DOTAP@Alloferon-1-Heparin/Protamine), 42

PTX/Alloferon-1 NP (PTX-DOTAP@Alloferon-1-Heparin/Protamine) (n=8). b) Tumor volume change in each group (n=8). c) Body weight change of B16-F10 bearing mice after intravenous injection of different formulations. Significance was measured by two-way ANOVA with Bonferroni post-test (n=8). d) Survival change after intravenous injection of different formulations. Significance was measured by log-rank test. (results were shown in mean ± S.D., ‘***’ indicates P < 0.001) (n=8). e) TUNEL staining (apoptotic cells shown in green) and statistical analysis of

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TUNEL positive ratio (n=3). f) Ki67 immunohistochemistry (proliferative cells shown in brown)

of tumor sections and Ki67 positive ratio (n=3). (results were shown in mean ± S.D., ‘*’ indicates P < 0.05, ‘**’ indicates P < 0.01 and ‘***’ indicates P < 0.001, all scale bars are 100 μm in

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TUNEL and Ki67 assay.)

Figure 7. Alloferon-1 activates the immune system that is inhibited by tumor cells in the tumor microenvironment. a) and b) Changes in the levels of IFN-γ and TNF-α in peripheral blood of mice after treatment in each formulation group. c) The difference in immunofluorescence intensity of IFN-γ and TNF-α in mouse tumor sections after treatment in each preparation group. d) and e) Differences in immunofluorescence of NKG2D and CD94 in mouse tumor sections after treatment 43

with each formulation group, and f) and g) their corresponding semi-quantitative data. h) The difference in immunofluorescence intensity of CD8 and granzyme B in mouse tumor sections after treatment in each formulation group. (n= 3, results were shown in mean ± S.D., ‘*’ indicates P <

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0.05, ‘**’ indicates P < 0.01 and ‘***’ indicates P < 0.001, all scale bars are 100 μm)

Figure 8. Histopathology study of mice treating with various groups. The sections of heart, liver, spleen, lung and kidney were stained with hematoxylin and eosin. Arrows indicate the lesion sites of liver, lung and kidney. (n=3, scale bars are 50 μm)

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