# Preparation of Cholesteryl-Modified Aminated Pullulan Nanoparticles to Evaluate Nanoparticle of Hydrophobic Degree on Drug Release and Cytotoxicity

Authors: Huabing Yuan; Wu Zhong; Rong Wang; Ping Zhou; Yu Nie; Wei Hu; Xiaojun Tao; Ping Yang
Journal: Journal of Nanomaterials
 (2020)

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## Abstract

The formation of nanoparticles (NPs) and surface properties such as size and charge are affected by the amphiphilic property of polymer, which is vital for evaluating their function. Here, we synthesized cholesteryl-modified aminated pullulan polymers (CHPNs) with different amounts of cholesterol succinate (CHS). We characterized the three hydrophobically modified polymers (CHPN1, CHPN2, and CHPN3) (CHS: Pu‐NH2=1/5,2/5,3/5) by Fourier transform infrared spectrometry. Dynamic light scattering (DLS) was used to measure particle size and zeta potential of CHPN NPs. The particle sizes of the three NPs CHPN1, CHPN2, and CHPN3 were 178.0, 144.4, and 97.8 nm, respectively. The particle size was related to the cholesteryl substitution of polymers to a certain extent: the stronger the hydrophobicity, the smaller the particle size. In 48 h, the drug release for CHPN3 and CHPN1 NPs was 57.8% and 72.7%. Thus, the NPs showed good sustained drug release: the greater the degree of hydrophobic substitution, the better the sustained release. The cytotoxicity findings were reversed: CHPN1 NPs, with low hydrophobic substitution, showed the best inhibition of Lewis lung cancer cells.


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## Body

## 1. Introduction

Amphiphilic polymers can spontaneously form shell-core structure nanoparticles (NPs) by self-assembly in the aqueous phase [1]. As an excellent drug carrier, NPs have been widely used in the field of antitumor drug delivery [2, 3]. Self-assembly of polymers to form NPs is aimed at maintaining a stable structure and morphology by hydrophobic interaction between hydrophobic groups and hydrogen bonding, electrostatic interaction, etc. [4, 5]. The hydrophobic interaction between the hydrophobic groups always has a leading role in the formation of NPs [6]. Therefore, many studies of polymer synthesis have examined the formation properties and function of NPs by hydrophobic substitution. Some of these studies show that changing the grafting density of the hydrophobic-substituted base can change the size, shape, elasticity, solubility, and stability of NPs [7–10].The effect of hydrophobic substitution degree on some properties of NPs will affect the function of the NPs. The greater the degree of hydrophobic substitution, the smaller the particle size of self-assembled NPs [10]. The size of NPs plays a decisive role in the accuracy of their passive targeted delivery of anticancer drugs: 50 to 200 nm NPs can make better use of the high permeability of blood vessels near the tumor and enhanced permeability retention (EPR) effect of passive targeting of tumor sites [11]; therefore, the effect of hydrophobic substitution on the size of NPs is of great significance. The particle size is also associated with body immunity [12]; indeed, with about100 nm particle size, phagocytosis of macrophages can be avoided [13, 14]. In addition, the greater the hydrophobic substitution, the more stable the particle and the longer the circulation in the body [15]; however, such NPs have lower deformability and more difficulty entering tumor cells than flexible hydrophobic particles [16]. Hydrophobic substitution also affects the release of carrier NPs and even the activity and effect of the loaded drug [17]. The hydrophobic part of NPs determines the immune response. Also, cytotoxicity is associated with the degree of hydrophobic substitution of NPs [18]. Therefore, the hydrophobic substitution degree in constructing drug carrier NPs should be carefully controlled, and the important properties and functional changes of NPs caused by different hydrophobic substitutions need investigation.Polysaccharides such as dextran, chitosan, and pullulan can be modified by hydrophobic groups such as long carbon chains or cholesterol groups to form amphiphilic polymers [19, 20]. Pullulan polysaccharide is nontoxic, water-soluble, biocompatible, and easy to biodegrade and contains many active hydroxyl groups [21]. It is easy to connect hydrophobic groups by chemical modification such as esterification to form amphiphilic graft copolymers [22]. Previous study of cholesterol-modified pullulan polysaccharide showed that it has high loading capacity and a sustained-release effect [23]. The hydrophobicity of NPs can be changed by changing the substitution degree of cholesterol, which changes the size and drug loading and release of NPs [24]. However, because of the large surface area and high surface activity, NPs can easily agglomerate, which will have adverse effects [10, 25]. Because of electrostatic repulsion, charged NPs are not easily aggregated as compared with uncharged NPs. Cell membranes are negatively charged, so they have high affinity with positive substances that will be highly uptaken by cells. The amino group, a positively charged and hydrophilic group, can be modified on the surface of drug-loaded NPs to make them positive, thereby increasing the internalization in cells [26].In this study, we synthesized amphiphilic polymers with different hydrophobic modifications, prepared blank NPs, and methotrexate- (MTX-) loaded NPs (CHMP NPs). To explore the effect of hydrophobic degree on the structure and function of NPs, we used dynamic light scattering (DLS) to observe the morphology, size, and potential of the assembled NPs as well as drug release and cytotoxicity of drug-loaded NPs in vitro.


## 2. Materials and Methods

### 2.1. Materials

Cholesterol and Boc-glycine were from Shanghai Ruiyong Biotechnology; pullulan, 4-dimethylaminoaniline (DMAP), 1-ethyl-(3-dimethylaminopropyl) carbodiimide salt, and acid salt (EDC) were from Aladdin Reagent (Shanghai); and succinic anhydride, dimethyl sulfoxide (DMSO), pyridine, and trifluoroacetic acid (TFA) were from Sinopharm Chemical Reagent. Double distilled water was homemade, and reagents were of analytical grade. Mouse Lewis lung carcinoma (LLC) cells were purchased from Shanghai GeneChem Co., LTD. High-glucose DMEM, phosphate-buffered saline (PBS), and fetal calf serum (10099-141) were from Gibco.


### 2.2. Synthesis of CHPN Polymer (Figure1)

Figure 1
Chemical synthesis of CHPN polymer.


#### 2.2.1. Synthesis of Succinic Anhydride Cholesterol (CHS)

NIn brief, 2 g cholesterol, and 1 g succinic acid were weighed into a round-bottom flask, and an appropriate amount of pyridine was added to dissolve them; the reaction was stirred for 48 h in an oil bath at 50°C. The reacted liquid was then poured into 1000 mL of 0°C double distilled water. Hydrochloric acid was added to adjust the pH to 4, and the beaker was chilled for 2 h and suction-filtered. The white solid was dissolved in 60 mL solution (ethanol : ethyl acetate 1 : 1), and succinic acid (CHS) was crystallized.


#### 2.2.2. Synthesis of Aminated Pullulan (Pu-NH2)

In brief, 10 g pullulan and 5 g Boc-glycine (0.03 mol) were added to 3.7 g DMAP (0.03 mol), dissolved in 200 mL dehydrated DMSO, and stirred at 50°C for 2 days; then, 30 mL TFA was added for 2 h incubation. After removing Boc-glycine, the mixture was added to 800 mL absolute ethanol and suction-filtered. The obtained solid was dissolved in water, filtered, and then dialyzed in a dialysis bag for 12 h and lyophilized with a freeze dryer to obtain a Pu-NH2 solid product.


#### 2.2.3. Synthesis of Different Hydrophobically Modified Aminated Pullulan Polymers (CHPNs)

Three similar reaction systems were selected, with 0.56 g Pu-NH2 dissolved in 10 mL DMSO. According to the molar ratio of CHS and sugar units 1/5, 2/5, and 3/5, namely, 0.2, 0.4, and 0.6 g CHS; 0.18 g DMAP (1.5 mmol); and 0.35 g EDC (1.8 mmol), the CHS was dissolved in 10 mL DMSO and stirred at room temperature to activate CHS, and the activated reaction liquid was dropped into the DMSO solution of amino pullulan polysaccharide to synthesize CHPN1, CHPN2, and CHPN3. In short, the content was mixed at 50°C with oil bath mixing and a heating reaction for 48 h. The reaction was stopped and cooled to room temperature; then, the reaction liquid was dropped into an appropriate amount of anhydrous ethanol, continuously stirred to precipitate the white precipitate, extracted, and filtered; then, an appropriate amount of anhydrous ethanol, tetrahydrofuran, and ether washing products was added, at 50°C dry under standby treatment, to produce a light yellow solid.


#### 2.2.4. Characterization of Polymers by Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR)

A small amount of solid succinic anhydride cholesterol, amino pullulan polysaccharide, and different CHPNs as well as infrared dry potassium bromide powder was mixed under infrared light and pressed into transparent sample potassium bromide tablets. The infrared spectra of samples were obtained by scanning in the range of 500~4000 cm-1. A small amount of solid succinic anhydride cholesterol ester, amino pullulan polysaccharide, and different CHPNs was dissolved in deuterium DMSO and analyzed by NMR. The degree of substitution of CHS was determined on the basis of the α-1,4 and α-1,6 glycosidic bonds and the area under the methylene peak [24]:
(1)DS=Aδ2.534Aδ5.05+Aδ4.68×100%.


#### 2.2.5. DSC and XRD of Active Pharmaceutical Ingredient (API) and Polymer

The differential scanning calorimetry (DSC) analysis was performed using a Differential Scanning Calorimeter (DSC200F3, NETZSCH, Germany); samples were heated from 10 to 200°C at a heating rate of 10°C/min under nitrogen atmosphere. X-ray diffraction (XRD) was performed using a diffractometer (D8 Discover, Bruker AXS, Germany), with an interval 0.02 s in the2θ range of 10°–90°.


### 2.3. Preparation of Blank and Drug-Loaded NPs

NPs were prepared by dialysis. Specifically, 0.3 g amino-modified CHPNs, CHPN1, CHPN2, and CHPN3, were dissolved in DMSO, stirred at room temperature until they completely dissolved, and then put into dialysis bags (7 kDa). After dialysis for 48 h with double distilled water, DMSO was removed to obtain hydrophobically modified amino pullulan polysaccharide NPs, CHPN1, CHPN2, and CHPN3 NPs. Drug-loading NPs were also formed by the dialysis method, taking CHPN1, CHPN2, and CHPN3, 0.3 g each, stirring them until completely dissolved, adding DMSO, then adding 2 mg of the model drug MTX dissolved in 2 mL of DMSO, and dropping them into the above solution, by using the dialysis bag (7 kDa intercept molecular weight) for dialysis in double distilled water for 24 h to remove organic solvent and free drug MTX, then to obtain CHMPN1, CHMPN2, and CHMPN3 NPs. The preparation of drug-loading NPs was in the dark at all times.


### 2.4. Characterization of Blank and Drug-Loaded CHPN NPs

#### 2.4.1. Dynamic Light Scattering

The prepared blank and drug-loaded NPs were placed into colorimetric cups, then placed into the DLS granulometer (DLS, Zetasizer 3000 HS, Malvern Instruments, Malvern, UK). Each sample was tested three times, 1 min/time. The test conditions were argon ion laser, wavelength 658 nm, temperature25±0.1degrees, and DLS angle 90°. The zeta potential was determined at the same time. The operating conditions were 11.4 v/cm, 13.0 mA, and 25°C. The sample solvent was diluted with distilled water.


#### 2.4.2. Transmission Electron Microscopy (TEM) and Scanning Electron Microscope (SEM)

Blank CHPN NPs (CHPN1 NPs) with the same substitution and drug-loaded NPs (CHPN1 NPs) were dropped on copper mesh coated with carbon support film, dyed with 2% phosphorus-uric acid, and morphology was observed by TEM after natural drying. The SEM images of CHPN NPs and CHMPN NPs were obtained using S-4800 instrument (Hitachi Ltd.) with an accelerating voltage of 10.0 kV.


### 2.5. Drug Loading and Release In Vitro

An amount of 10 mg drug-loaded NPs was accurately weighed, dispersed in 5 mL of 10 mmol/L phosphate buffer solution, sonicated for 10 min, and filtered through a 0.2μm microporous membrane, and the filtrate was topped up to 10 mL. An amount of 2 mL solution was pipetted into a cuvette, and absorbance was measured at 303 nm by using a microplate spectrophotometer (UV-384 plus, Molecular Devices, Thermo Fisher Scientific, Waltham, MA, USA), with reference to MTX standards. The curve was used to determine the concentration of MTX in the solution, and drug loading (LC) of different nanocarriers of MTX was calculated as follows:
(2)LC%=amountofdrugintheNPNPweight×100%.Drug release was measured by the dialysis method. Briefly, 5 mg drug-loaded NPs were dispersed in 10 mL phosphate buffer (pH 7.4, temperature 37.5°C) and loaded into a dialysis bag (MW=8‐12kDa) and separated at 25 mL, pH 7.4. Dialysis was carried out in PBS (release medium) at 37°C and shaking at 80 rpm. Then, 2 mL release medium was collected for sampling and replaced with an equal volume of fresh solution at predetermined time intervals (Tn=0,0.5,1,2,4,8,12,24,48,72h). The absorbance of MTX in the solution was measured by using a microplate spectrophotometer, and three samples were taken from each group to determine the released MTX. The percentage of drug release (Q%) was calculated as follows:
(3)Q%=Cn×V+Vn∑t=0nCiWNP×LC%.where W is the NP weight, Cn is the sample concentration at Tn, V is the total volume of release medium, Vn is the sample volume (2 mL), and Ci is the sample concentration at Ti (i=0,0.5,1,⋯,nhours, both V0 and C0 are equal to zero).


### 2.6. Cell Culture

Mouse Lewis lung carcinoma (LLC) cells were cultured with 13% fetal bovine serum, 1% double antibody (penicillin, streptomycin), inoculated in a 25 cm2 culture flask, and cultured in a 37°C incubator at 5% CO2 for amplification. The experimental cells were all derived from logarithmic growth-phase cells.


### 2.7. In Vitro Cytotoxicity

An amount of 3000 mouse lung cancer LLC cells was placed per well in a 96-well plate. After cells attached, CHMPN1, CHMPN2, CHMPN3 NPs and free MTX with the same MTX concentration were added. The concentrations were10, 20, 30, 40, and 50 μg/mL. After the drug was added for 24 or 48 h, 10 mL CCK-8 reagent was added and incubated for 2 h. Then, absorbance at 450 nm wavelength was detected by using an enzyme-labeling instrument. A group without the drug was a control, and a blank cell group was a control. The inhibition rate of different drug preparations for LCC cells was calculated as follows:
(4)Inhibitionrate%=ODexperimentalgroup−ODblankgroupODcontrolgroup−ODblankgroup×100%.


### 2.8. Statistical Analysis

Statistical analysis was performed with origin 8.5 and GraphPad Prism 7. Every cell experimental treatment was repeated independently for at least three times, and all of the results were expressed as mean with 95% CI.


## 2.1. Materials

Cholesterol and Boc-glycine were from Shanghai Ruiyong Biotechnology; pullulan, 4-dimethylaminoaniline (DMAP), 1-ethyl-(3-dimethylaminopropyl) carbodiimide salt, and acid salt (EDC) were from Aladdin Reagent (Shanghai); and succinic anhydride, dimethyl sulfoxide (DMSO), pyridine, and trifluoroacetic acid (TFA) were from Sinopharm Chemical Reagent. Double distilled water was homemade, and reagents were of analytical grade. Mouse Lewis lung carcinoma (LLC) cells were purchased from Shanghai GeneChem Co., LTD. High-glucose DMEM, phosphate-buffered saline (PBS), and fetal calf serum (10099-141) were from Gibco.


## 2.2. Synthesis of CHPN Polymer (Figure1)

Figure 1
Chemical synthesis of CHPN polymer.


### 2.2.1. Synthesis of Succinic Anhydride Cholesterol (CHS)

NIn brief, 2 g cholesterol, and 1 g succinic acid were weighed into a round-bottom flask, and an appropriate amount of pyridine was added to dissolve them; the reaction was stirred for 48 h in an oil bath at 50°C. The reacted liquid was then poured into 1000 mL of 0°C double distilled water. Hydrochloric acid was added to adjust the pH to 4, and the beaker was chilled for 2 h and suction-filtered. The white solid was dissolved in 60 mL solution (ethanol : ethyl acetate 1 : 1), and succinic acid (CHS) was crystallized.


### 2.2.2. Synthesis of Aminated Pullulan (Pu-NH2)

In brief, 10 g pullulan and 5 g Boc-glycine (0.03 mol) were added to 3.7 g DMAP (0.03 mol), dissolved in 200 mL dehydrated DMSO, and stirred at 50°C for 2 days; then, 30 mL TFA was added for 2 h incubation. After removing Boc-glycine, the mixture was added to 800 mL absolute ethanol and suction-filtered. The obtained solid was dissolved in water, filtered, and then dialyzed in a dialysis bag for 12 h and lyophilized with a freeze dryer to obtain a Pu-NH2 solid product.


### 2.2.3. Synthesis of Different Hydrophobically Modified Aminated Pullulan Polymers (CHPNs)

Three similar reaction systems were selected, with 0.56 g Pu-NH2 dissolved in 10 mL DMSO. According to the molar ratio of CHS and sugar units 1/5, 2/5, and 3/5, namely, 0.2, 0.4, and 0.6 g CHS; 0.18 g DMAP (1.5 mmol); and 0.35 g EDC (1.8 mmol), the CHS was dissolved in 10 mL DMSO and stirred at room temperature to activate CHS, and the activated reaction liquid was dropped into the DMSO solution of amino pullulan polysaccharide to synthesize CHPN1, CHPN2, and CHPN3. In short, the content was mixed at 50°C with oil bath mixing and a heating reaction for 48 h. The reaction was stopped and cooled to room temperature; then, the reaction liquid was dropped into an appropriate amount of anhydrous ethanol, continuously stirred to precipitate the white precipitate, extracted, and filtered; then, an appropriate amount of anhydrous ethanol, tetrahydrofuran, and ether washing products was added, at 50°C dry under standby treatment, to produce a light yellow solid.


### 2.2.4. Characterization of Polymers by Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR)

A small amount of solid succinic anhydride cholesterol, amino pullulan polysaccharide, and different CHPNs as well as infrared dry potassium bromide powder was mixed under infrared light and pressed into transparent sample potassium bromide tablets. The infrared spectra of samples were obtained by scanning in the range of 500~4000 cm-1. A small amount of solid succinic anhydride cholesterol ester, amino pullulan polysaccharide, and different CHPNs was dissolved in deuterium DMSO and analyzed by NMR. The degree of substitution of CHS was determined on the basis of the α-1,4 and α-1,6 glycosidic bonds and the area under the methylene peak [24]:
(1)DS=Aδ2.534Aδ5.05+Aδ4.68×100%.


### 2.2.5. DSC and XRD of Active Pharmaceutical Ingredient (API) and Polymer

The differential scanning calorimetry (DSC) analysis was performed using a Differential Scanning Calorimeter (DSC200F3, NETZSCH, Germany); samples were heated from 10 to 200°C at a heating rate of 10°C/min under nitrogen atmosphere. X-ray diffraction (XRD) was performed using a diffractometer (D8 Discover, Bruker AXS, Germany), with an interval 0.02 s in the2θ range of 10°–90°.


## 2.2.1. Synthesis of Succinic Anhydride Cholesterol (CHS)

NIn brief, 2 g cholesterol, and 1 g succinic acid were weighed into a round-bottom flask, and an appropriate amount of pyridine was added to dissolve them; the reaction was stirred for 48 h in an oil bath at 50°C. The reacted liquid was then poured into 1000 mL of 0°C double distilled water. Hydrochloric acid was added to adjust the pH to 4, and the beaker was chilled for 2 h and suction-filtered. The white solid was dissolved in 60 mL solution (ethanol : ethyl acetate 1 : 1), and succinic acid (CHS) was crystallized.


## 2.2.2. Synthesis of Aminated Pullulan (Pu-NH2)

In brief, 10 g pullulan and 5 g Boc-glycine (0.03 mol) were added to 3.7 g DMAP (0.03 mol), dissolved in 200 mL dehydrated DMSO, and stirred at 50°C for 2 days; then, 30 mL TFA was added for 2 h incubation. After removing Boc-glycine, the mixture was added to 800 mL absolute ethanol and suction-filtered. The obtained solid was dissolved in water, filtered, and then dialyzed in a dialysis bag for 12 h and lyophilized with a freeze dryer to obtain a Pu-NH2 solid product.


## 2.2.3. Synthesis of Different Hydrophobically Modified Aminated Pullulan Polymers (CHPNs)

Three similar reaction systems were selected, with 0.56 g Pu-NH2 dissolved in 10 mL DMSO. According to the molar ratio of CHS and sugar units 1/5, 2/5, and 3/5, namely, 0.2, 0.4, and 0.6 g CHS; 0.18 g DMAP (1.5 mmol); and 0.35 g EDC (1.8 mmol), the CHS was dissolved in 10 mL DMSO and stirred at room temperature to activate CHS, and the activated reaction liquid was dropped into the DMSO solution of amino pullulan polysaccharide to synthesize CHPN1, CHPN2, and CHPN3. In short, the content was mixed at 50°C with oil bath mixing and a heating reaction for 48 h. The reaction was stopped and cooled to room temperature; then, the reaction liquid was dropped into an appropriate amount of anhydrous ethanol, continuously stirred to precipitate the white precipitate, extracted, and filtered; then, an appropriate amount of anhydrous ethanol, tetrahydrofuran, and ether washing products was added, at 50°C dry under standby treatment, to produce a light yellow solid.


## 2.2.4. Characterization of Polymers by Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR)

A small amount of solid succinic anhydride cholesterol, amino pullulan polysaccharide, and different CHPNs as well as infrared dry potassium bromide powder was mixed under infrared light and pressed into transparent sample potassium bromide tablets. The infrared spectra of samples were obtained by scanning in the range of 500~4000 cm-1. A small amount of solid succinic anhydride cholesterol ester, amino pullulan polysaccharide, and different CHPNs was dissolved in deuterium DMSO and analyzed by NMR. The degree of substitution of CHS was determined on the basis of the α-1,4 and α-1,6 glycosidic bonds and the area under the methylene peak [24]:
(1)DS=Aδ2.534Aδ5.05+Aδ4.68×100%.


## 2.2.5. DSC and XRD of Active Pharmaceutical Ingredient (API) and Polymer

The differential scanning calorimetry (DSC) analysis was performed using a Differential Scanning Calorimeter (DSC200F3, NETZSCH, Germany); samples were heated from 10 to 200°C at a heating rate of 10°C/min under nitrogen atmosphere. X-ray diffraction (XRD) was performed using a diffractometer (D8 Discover, Bruker AXS, Germany), with an interval 0.02 s in the2θ range of 10°–90°.


## 2.3. Preparation of Blank and Drug-Loaded NPs

NPs were prepared by dialysis. Specifically, 0.3 g amino-modified CHPNs, CHPN1, CHPN2, and CHPN3, were dissolved in DMSO, stirred at room temperature until they completely dissolved, and then put into dialysis bags (7 kDa). After dialysis for 48 h with double distilled water, DMSO was removed to obtain hydrophobically modified amino pullulan polysaccharide NPs, CHPN1, CHPN2, and CHPN3 NPs. Drug-loading NPs were also formed by the dialysis method, taking CHPN1, CHPN2, and CHPN3, 0.3 g each, stirring them until completely dissolved, adding DMSO, then adding 2 mg of the model drug MTX dissolved in 2 mL of DMSO, and dropping them into the above solution, by using the dialysis bag (7 kDa intercept molecular weight) for dialysis in double distilled water for 24 h to remove organic solvent and free drug MTX, then to obtain CHMPN1, CHMPN2, and CHMPN3 NPs. The preparation of drug-loading NPs was in the dark at all times.


## 2.4. Characterization of Blank and Drug-Loaded CHPN NPs

### 2.4.1. Dynamic Light Scattering

The prepared blank and drug-loaded NPs were placed into colorimetric cups, then placed into the DLS granulometer (DLS, Zetasizer 3000 HS, Malvern Instruments, Malvern, UK). Each sample was tested three times, 1 min/time. The test conditions were argon ion laser, wavelength 658 nm, temperature25±0.1degrees, and DLS angle 90°. The zeta potential was determined at the same time. The operating conditions were 11.4 v/cm, 13.0 mA, and 25°C. The sample solvent was diluted with distilled water.


### 2.4.2. Transmission Electron Microscopy (TEM) and Scanning Electron Microscope (SEM)

Blank CHPN NPs (CHPN1 NPs) with the same substitution and drug-loaded NPs (CHPN1 NPs) were dropped on copper mesh coated with carbon support film, dyed with 2% phosphorus-uric acid, and morphology was observed by TEM after natural drying. The SEM images of CHPN NPs and CHMPN NPs were obtained using S-4800 instrument (Hitachi Ltd.) with an accelerating voltage of 10.0 kV.


## 2.4.1. Dynamic Light Scattering

The prepared blank and drug-loaded NPs were placed into colorimetric cups, then placed into the DLS granulometer (DLS, Zetasizer 3000 HS, Malvern Instruments, Malvern, UK). Each sample was tested three times, 1 min/time. The test conditions were argon ion laser, wavelength 658 nm, temperature25±0.1degrees, and DLS angle 90°. The zeta potential was determined at the same time. The operating conditions were 11.4 v/cm, 13.0 mA, and 25°C. The sample solvent was diluted with distilled water.


## 2.4.2. Transmission Electron Microscopy (TEM) and Scanning Electron Microscope (SEM)

Blank CHPN NPs (CHPN1 NPs) with the same substitution and drug-loaded NPs (CHPN1 NPs) were dropped on copper mesh coated with carbon support film, dyed with 2% phosphorus-uric acid, and morphology was observed by TEM after natural drying. The SEM images of CHPN NPs and CHMPN NPs were obtained using S-4800 instrument (Hitachi Ltd.) with an accelerating voltage of 10.0 kV.


## 2.5. Drug Loading and Release In Vitro

An amount of 10 mg drug-loaded NPs was accurately weighed, dispersed in 5 mL of 10 mmol/L phosphate buffer solution, sonicated for 10 min, and filtered through a 0.2μm microporous membrane, and the filtrate was topped up to 10 mL. An amount of 2 mL solution was pipetted into a cuvette, and absorbance was measured at 303 nm by using a microplate spectrophotometer (UV-384 plus, Molecular Devices, Thermo Fisher Scientific, Waltham, MA, USA), with reference to MTX standards. The curve was used to determine the concentration of MTX in the solution, and drug loading (LC) of different nanocarriers of MTX was calculated as follows:
(2)LC%=amountofdrugintheNPNPweight×100%.Drug release was measured by the dialysis method. Briefly, 5 mg drug-loaded NPs were dispersed in 10 mL phosphate buffer (pH 7.4, temperature 37.5°C) and loaded into a dialysis bag (MW=8‐12kDa) and separated at 25 mL, pH 7.4. Dialysis was carried out in PBS (release medium) at 37°C and shaking at 80 rpm. Then, 2 mL release medium was collected for sampling and replaced with an equal volume of fresh solution at predetermined time intervals (Tn=0,0.5,1,2,4,8,12,24,48,72h). The absorbance of MTX in the solution was measured by using a microplate spectrophotometer, and three samples were taken from each group to determine the released MTX. The percentage of drug release (Q%) was calculated as follows:
(3)Q%=Cn×V+Vn∑t=0nCiWNP×LC%.where W is the NP weight, Cn is the sample concentration at Tn, V is the total volume of release medium, Vn is the sample volume (2 mL), and Ci is the sample concentration at Ti (i=0,0.5,1,⋯,nhours, both V0 and C0 are equal to zero).


## 2.6. Cell Culture

Mouse Lewis lung carcinoma (LLC) cells were cultured with 13% fetal bovine serum, 1% double antibody (penicillin, streptomycin), inoculated in a 25 cm2 culture flask, and cultured in a 37°C incubator at 5% CO2 for amplification. The experimental cells were all derived from logarithmic growth-phase cells.


## 2.7. In Vitro Cytotoxicity

An amount of 3000 mouse lung cancer LLC cells was placed per well in a 96-well plate. After cells attached, CHMPN1, CHMPN2, CHMPN3 NPs and free MTX with the same MTX concentration were added. The concentrations were10, 20, 30, 40, and 50 μg/mL. After the drug was added for 24 or 48 h, 10 mL CCK-8 reagent was added and incubated for 2 h. Then, absorbance at 450 nm wavelength was detected by using an enzyme-labeling instrument. A group without the drug was a control, and a blank cell group was a control. The inhibition rate of different drug preparations for LCC cells was calculated as follows:
(4)Inhibitionrate%=ODexperimentalgroup−ODblankgroupODcontrolgroup−ODblankgroup×100%.


## 2.8. Statistical Analysis

Statistical analysis was performed with origin 8.5 and GraphPad Prism 7. Every cell experimental treatment was repeated independently for at least three times, and all of the results were expressed as mean with 95% CI.


## 3. Result

### 3.1. FTIR and1H NMR Analysis

The synthesis of polymer CHPN, esterified from CHS and Pu-NH2, is in Figure 1. The structural characterization of the polymer was proved by FTIR and NMR analysis. The peak near 1167-1069 cm−1 is the C-N stretching vibration peak, the peak near 1500 cm−1 is the N-H and C-N coupling vibration peak, and the peak near 1730 cm−1 (C=O stretching vibration peak) proves the successful grafting of NH2 on pullulan (Figure 2(b)).Figure 2
FTIR spectra for cholesterol succinate (CHS) (a), Pu-NH2 (b), CHPN1 (c), CHPN2 (d), and CHPN3 (e).As compared with CHS, Pu-NH2 and CHPN have stronger -OH characteristic peaks near 3450 cm-1 because they contain multiple hydroxyl groups, and -NH also has a characteristic peak near 3400 cm-1. The superimposition of the two enhances the absorption. However, CHS is attached to the polysaccharide hydroxyl group when CHPN is synthesized, and the strength of the hydroxyl group gradually decreases. The decrease is more pronounced with increasing amount of CHS because the number of hydroxyl groups decreases as ester bonds are formed between the two. The stretching vibration peaks of methyl and methylene were close to 2900 cm-1, but the absorption peaks of CHPN and CHS were significantly enhanced close to 2900 cm-1, and an obvious ester carbonyl absorption peak appeared close to1750-1710 cm-1. The CHS absorption peak at 1167 cm-1 represented a C-O stretching vibration peak, and the strong and broad peaks of Pu-NH2 and CHPN near this represented a C-O-C stretching vibration peak. Their fingerprint regions are approximately similar, indicating structural similarities.The characteristic NMR spectra peaks for CHS are in Figure3: 0.8-1.5 ppm is the side chain -CH2 and ring -CH2, 2.5-3.5 ppm is the characteristic peak of CH2 (-CH2CO) next to its ester bond, 4.3 ppm is the characteristic peak of its double bond -C=CH, and for Pu-NH2, 1.0 ppm is the alkyl hydrogen on the sugar ring. The characteristic peaks for -OH and -CH2CO are 3-4 ppm, and those for -CH2CO and -OH are 4.6-5.5 ppm. As compared with CHS and Pu-NH2, both CHPN spectra combined the absorption characteristics of CHS and Pu-NH2. CH=CH2 peaks of CHS were found near 4.3 ppm, and the unique -CH2CO and -OH peaks for Pu-NH2 were also found at 4.6-5.5 ppm. Thus, the successful synthesis of CHPN can be demonstrated by the chemical binding of cholesterol to the amino pullulan long chain. Calculated according to the formula, the cholesterol substitution degree of CHPN1, CHPN2, CHPN3 was 3.56%, 5.68% and 6.74%, respectively.Figure 3
1H NMR spectra for CHS, Pu-NH2, CHPN1, CHPN2, and CHPN3.
(a)
(b)


### 3.2. DSC and XRD of CHS, Pu-NH2, and Polymer

As shown in Figures4(a)–4(c), CHS was crystalline at room temperature, while Pu-NH2 and their polymer CHPN were not. The XRD indicated the change in crystallinity of CHS when it interacted with Pu-NH2. And the DSC was shown in Figure 4(d). The melting peak of CHS was at 179.5°C and Pu-NH2 was at 125.9°C. There was no endothermic peak of cholesterol in the polymer, suggesting its good compatibility.Figure 4
XRD (a–c) and standard DSC curves (d) of the APIs and polymer CHPN.
(a)
(b)
(c)
(d)


### 3.3. Size Distribution, Potential, and Electron Microscopy Analysis of Blank NPs

The mean particle size and dispersion of CHPN1, CHPN2, and CHPN3 NPs were 178.0±4.2nm and 0.257±0.048nm, 144.4±3.6nm and 0.199±0.036nm, and 97.8±2.2nm and 0.196±0.024nm, respectively. Therefore, the particle size of NPs with different hydrophobic substitution degrees greatly differed, and the particle size of CHS/Pu-NH2 with high hydrophobic substitution was small. The dispersion of NPs also decreased with increasing hydrophobic substitution degree, which indicates that hydrophobic force plays an effective role in the stability of CHPN NPs; furthermore, hydrophobic action is the driving force of CHPN NP self-assembly. The mean zeta potentials for CHPN1, CHPN2, and CHPN3 NPs were 4.29±0.94, 3.39±0.77, and 2.56±0.18mV, respectively. (Table 1) The zeta potential difference was small, and potentials were positive. And the positive potential was smaller with higher degree of substitution. TEM and SEM showed CHPN NPs with a regular spherical structure.Table 1
Size, polydispersity index (PDI), and zeta potential of CHPN NPs.
NPs
Size (nm)
PDI (nm)
Zeta potential (mV)
CHPN1 NPs
178.0±4.2
0.257±0.048
4.29±0.94
CHPN2 NPs
144.4±3.6
0.199±0.036
3.39±0.77
CHPN3 NPs
97.8±2.2
0.196±0.024
2.56±0.18


### 3.4. Analysis of Drug-Loaded NPs

The mean size and dispersion of the drug MTX loadings of CHPN1 NPs, CHPN2 NPs, and CHPN3 NPs were 195.9±4.6nm and 0.338±0.065, 158.3±3.8nm and 0.302±0.057, and 123.2±2.9nm and 0.246±0.046, respectively. The zeta potentials were 3.46±0.27, 3.16±0.62, and 1.99±0.80mV, respectively. The change rule for all three was the same as for the blank nanometer but decreased with hydrophobic substitution degree. CHPN1 and drug-loaded CHPN1 NPs had a regular spherical structure (Figures 5 and 6). The drug loading proportion for CHPN1, CHPN2, and CHPN3 NPs was 5.2±0.1%, 6.7±0.4%, and 8.6±0.3%, respectively. NPs with high hydrophobic substitution degree had higher drug loading. Because of the negative charge of MTX [27], CHPN NPs were mainly affected by hydrophobic and electrostatic forces when carrying drugs, and the drug loading of CHPN NPs was positively related to the degree of hydrophobic substitution.Figure 5
Size distribution and zeta potentials of CHPN1 NPs (A0), CHPN2 NPs (B0), and CHPN3 NPs (C0) (a, b). TEM (c) and SEM (d) image of CHPN1 NPs.
(a)
(b)
(c)
(d)Figure 6
The size distributions and zeta potential of CHPN1 NPs (A), CHPN2 NPs (B), and CHPN3 NPs (C) after loading drugs MTX (a, b). TEM (c) and SEM (d) image of CHPN1 NPs after loading drugs MTX.
(a)
(b)
(c)
(d)


### 3.5. Drug Release In Vitro

As shown in Figure7, the release of free drugs was relatively rapid, releasing 80.5% within 12 h, while the drugs from NPs exhibited two phases of releasing. The two phases included a rapid release in the first 12 h and a sustained release followed. The rapid releasing was related to the surface-absorbed MTX, and the slowly sustained releasing was probably related to the encapsulated MTX. CHPN3 NPs, with a high degree of hydrophobic substitution, released 57.8% of drugs. CHPN2 NPs released 64.4% in 48 h. CHPN1 NPs, with the lowest degree of substitution, released 72.7% within 48 h. The release test indicated that NPs with high hydrophobic substitution had slower drug release; the higher the hydrophobic degree, the stronger the slow-release effect.Figure 7
Methotrexate (MTX) release from different NPs or free MTX.


### 3.6. In Vitro Cytotoxicity of Drug-Loaded NPs

To confirm the inhibitory effect of MTX-loaded NPs on cancer cells and compare the function of different hydrophobically substituted NPs, we used mouse LLC cells, a lung carcinoma cell line, for cell viability testing. MTX is a broad-spectrum anticancer drug in clinical practice. Different hydrophobic NPs were used to encapsulate equal amounts of free MTX, for different preparations. The cell viability ratio with treatment for 24 and 48 h (Figures8 and 9) was in the order of CHMPN1 NPs<CHMPN2 NPs<MTX<CHMPN3 NPs. The cell viability of LLC cells under the inhibition of MTX, CHMPN1, CHMPN2, and CHMPN3 NPs for 24 h decreased to 61.8%, 47.1%, 57.2%, and 65.7%, respectively. The inhibition with 48 h decreased more, with a consistent cytotoxicity trend. The average IC50 for preparations are in Table 2. The cytotoxicity of CHMPN1 and CHMPN2 NPs with low CHS/Pu-NH2 substitution was high, even better than that with the free drug MTX. CHMPN3 NP toxicity in LLC cells was relatively less and slightly less than free drug.Figure 8
Cell viability with different preparations at 24 h.Figure 9
Cell viability with different preparations at 48 h.Table 2
IC50 of NPs and free MTX at 24 and 48 h.
Sample
IC50 (μg/mL) at
24 h
48 h
Free MTX
101.9
17.10
CHMPN1 NPs
51.38
13.06
CHMPN2 NPs
73.70
15.18
CHMPN3 NPs
132.70
19.09


## 3.1. FTIR and1H NMR Analysis

The synthesis of polymer CHPN, esterified from CHS and Pu-NH2, is in Figure 1. The structural characterization of the polymer was proved by FTIR and NMR analysis. The peak near 1167-1069 cm−1 is the C-N stretching vibration peak, the peak near 1500 cm−1 is the N-H and C-N coupling vibration peak, and the peak near 1730 cm−1 (C=O stretching vibration peak) proves the successful grafting of NH2 on pullulan (Figure 2(b)).Figure 2
FTIR spectra for cholesterol succinate (CHS) (a), Pu-NH2 (b), CHPN1 (c), CHPN2 (d), and CHPN3 (e).As compared with CHS, Pu-NH2 and CHPN have stronger -OH characteristic peaks near 3450 cm-1 because they contain multiple hydroxyl groups, and -NH also has a characteristic peak near 3400 cm-1. The superimposition of the two enhances the absorption. However, CHS is attached to the polysaccharide hydroxyl group when CHPN is synthesized, and the strength of the hydroxyl group gradually decreases. The decrease is more pronounced with increasing amount of CHS because the number of hydroxyl groups decreases as ester bonds are formed between the two. The stretching vibration peaks of methyl and methylene were close to 2900 cm-1, but the absorption peaks of CHPN and CHS were significantly enhanced close to 2900 cm-1, and an obvious ester carbonyl absorption peak appeared close to1750-1710 cm-1. The CHS absorption peak at 1167 cm-1 represented a C-O stretching vibration peak, and the strong and broad peaks of Pu-NH2 and CHPN near this represented a C-O-C stretching vibration peak. Their fingerprint regions are approximately similar, indicating structural similarities.The characteristic NMR spectra peaks for CHS are in Figure3: 0.8-1.5 ppm is the side chain -CH2 and ring -CH2, 2.5-3.5 ppm is the characteristic peak of CH2 (-CH2CO) next to its ester bond, 4.3 ppm is the characteristic peak of its double bond -C=CH, and for Pu-NH2, 1.0 ppm is the alkyl hydrogen on the sugar ring. The characteristic peaks for -OH and -CH2CO are 3-4 ppm, and those for -CH2CO and -OH are 4.6-5.5 ppm. As compared with CHS and Pu-NH2, both CHPN spectra combined the absorption characteristics of CHS and Pu-NH2. CH=CH2 peaks of CHS were found near 4.3 ppm, and the unique -CH2CO and -OH peaks for Pu-NH2 were also found at 4.6-5.5 ppm. Thus, the successful synthesis of CHPN can be demonstrated by the chemical binding of cholesterol to the amino pullulan long chain. Calculated according to the formula, the cholesterol substitution degree of CHPN1, CHPN2, CHPN3 was 3.56%, 5.68% and 6.74%, respectively.Figure 3
1H NMR spectra for CHS, Pu-NH2, CHPN1, CHPN2, and CHPN3.
(a)
(b)


## 3.2. DSC and XRD of CHS, Pu-NH2, and Polymer

As shown in Figures4(a)–4(c), CHS was crystalline at room temperature, while Pu-NH2 and their polymer CHPN were not. The XRD indicated the change in crystallinity of CHS when it interacted with Pu-NH2. And the DSC was shown in Figure 4(d). The melting peak of CHS was at 179.5°C and Pu-NH2 was at 125.9°C. There was no endothermic peak of cholesterol in the polymer, suggesting its good compatibility.Figure 4
XRD (a–c) and standard DSC curves (d) of the APIs and polymer CHPN.
(a)
(b)
(c)
(d)


## 3.3. Size Distribution, Potential, and Electron Microscopy Analysis of Blank NPs

The mean particle size and dispersion of CHPN1, CHPN2, and CHPN3 NPs were 178.0±4.2nm and 0.257±0.048nm, 144.4±3.6nm and 0.199±0.036nm, and 97.8±2.2nm and 0.196±0.024nm, respectively. Therefore, the particle size of NPs with different hydrophobic substitution degrees greatly differed, and the particle size of CHS/Pu-NH2 with high hydrophobic substitution was small. The dispersion of NPs also decreased with increasing hydrophobic substitution degree, which indicates that hydrophobic force plays an effective role in the stability of CHPN NPs; furthermore, hydrophobic action is the driving force of CHPN NP self-assembly. The mean zeta potentials for CHPN1, CHPN2, and CHPN3 NPs were 4.29±0.94, 3.39±0.77, and 2.56±0.18mV, respectively. (Table 1) The zeta potential difference was small, and potentials were positive. And the positive potential was smaller with higher degree of substitution. TEM and SEM showed CHPN NPs with a regular spherical structure.Table 1
Size, polydispersity index (PDI), and zeta potential of CHPN NPs.
NPs
Size (nm)
PDI (nm)
Zeta potential (mV)
CHPN1 NPs
178.0±4.2
0.257±0.048
4.29±0.94
CHPN2 NPs
144.4±3.6
0.199±0.036
3.39±0.77
CHPN3 NPs
97.8±2.2
0.196±0.024
2.56±0.18


## 3.4. Analysis of Drug-Loaded NPs

The mean size and dispersion of the drug MTX loadings of CHPN1 NPs, CHPN2 NPs, and CHPN3 NPs were 195.9±4.6nm and 0.338±0.065, 158.3±3.8nm and 0.302±0.057, and 123.2±2.9nm and 0.246±0.046, respectively. The zeta potentials were 3.46±0.27, 3.16±0.62, and 1.99±0.80mV, respectively. The change rule for all three was the same as for the blank nanometer but decreased with hydrophobic substitution degree. CHPN1 and drug-loaded CHPN1 NPs had a regular spherical structure (Figures 5 and 6). The drug loading proportion for CHPN1, CHPN2, and CHPN3 NPs was 5.2±0.1%, 6.7±0.4%, and 8.6±0.3%, respectively. NPs with high hydrophobic substitution degree had higher drug loading. Because of the negative charge of MTX [27], CHPN NPs were mainly affected by hydrophobic and electrostatic forces when carrying drugs, and the drug loading of CHPN NPs was positively related to the degree of hydrophobic substitution.Figure 5
Size distribution and zeta potentials of CHPN1 NPs (A0), CHPN2 NPs (B0), and CHPN3 NPs (C0) (a, b). TEM (c) and SEM (d) image of CHPN1 NPs.
(a)
(b)
(c)
(d)Figure 6
The size distributions and zeta potential of CHPN1 NPs (A), CHPN2 NPs (B), and CHPN3 NPs (C) after loading drugs MTX (a, b). TEM (c) and SEM (d) image of CHPN1 NPs after loading drugs MTX.
(a)
(b)
(c)
(d)


## 3.5. Drug Release In Vitro

As shown in Figure7, the release of free drugs was relatively rapid, releasing 80.5% within 12 h, while the drugs from NPs exhibited two phases of releasing. The two phases included a rapid release in the first 12 h and a sustained release followed. The rapid releasing was related to the surface-absorbed MTX, and the slowly sustained releasing was probably related to the encapsulated MTX. CHPN3 NPs, with a high degree of hydrophobic substitution, released 57.8% of drugs. CHPN2 NPs released 64.4% in 48 h. CHPN1 NPs, with the lowest degree of substitution, released 72.7% within 48 h. The release test indicated that NPs with high hydrophobic substitution had slower drug release; the higher the hydrophobic degree, the stronger the slow-release effect.Figure 7
Methotrexate (MTX) release from different NPs or free MTX.


## 3.6. In Vitro Cytotoxicity of Drug-Loaded NPs

To confirm the inhibitory effect of MTX-loaded NPs on cancer cells and compare the function of different hydrophobically substituted NPs, we used mouse LLC cells, a lung carcinoma cell line, for cell viability testing. MTX is a broad-spectrum anticancer drug in clinical practice. Different hydrophobic NPs were used to encapsulate equal amounts of free MTX, for different preparations. The cell viability ratio with treatment for 24 and 48 h (Figures8 and 9) was in the order of CHMPN1 NPs<CHMPN2 NPs<MTX<CHMPN3 NPs. The cell viability of LLC cells under the inhibition of MTX, CHMPN1, CHMPN2, and CHMPN3 NPs for 24 h decreased to 61.8%, 47.1%, 57.2%, and 65.7%, respectively. The inhibition with 48 h decreased more, with a consistent cytotoxicity trend. The average IC50 for preparations are in Table 2. The cytotoxicity of CHMPN1 and CHMPN2 NPs with low CHS/Pu-NH2 substitution was high, even better than that with the free drug MTX. CHMPN3 NP toxicity in LLC cells was relatively less and slightly less than free drug.Figure 8
Cell viability with different preparations at 24 h.Figure 9
Cell viability with different preparations at 48 h.Table 2
IC50 of NPs and free MTX at 24 and 48 h.
Sample
IC50 (μg/mL) at
24 h
48 h
Free MTX
101.9
17.10
CHMPN1 NPs
51.38
13.06
CHMPN2 NPs
73.70
15.18
CHMPN3 NPs
132.70
19.09


## 4. Discussion

The formation of NPs and surface properties such as size and charge are affected by the amphiphilic property of polymer, which is vital for evaluating their function [28, 29]. Wei et al. found that the increase in hydrophobicity substitution reduces the size of hyaluronic acid-deoxycholate self-assembled NPs and increases their drug encapsulation efficiency [30]. Li et al. found that the cellular uptake increased in the bigger nanoparticles [31]. Sun et al. reported that insertion of hydrophobic octanethiol onto the surface of 2 nm zwitterionic luminescent glutathione-coated gold NPs enhanced their affinity for cell membranes and increased cell uptake by more than an order of magnitude [32]. These studies demonstrate the diversity and heterogeneity of hydrophobic-induced NP functions and provide new avenues for promoting cellular uptake by NPs. However, too much hydrophobic substitution results in lower blood compatibility [33], causing NPs to be unstable in the body; too little hydrophobic substitution will cause too-high critical aggregation concentration [34], and aggregation to form NPs will not be easy. Therefore, suitable hydrophobically substituted NPs must be screened by experiments to optimize drug delivery and increase the absorption of drugs by tumor cells.In a previous study [23, 24], the drug loading of CHP NPs depended on the degree of cholesterol substitution in the polymer, and after reaching the critical concentration of aggregation to form NPs, the drug loading increased with increasing degree of substitution. Amino NPs had higher inhibitory activity in cancer cells than the two other kinds of NPs by grafting cholesterol without modification and the carboxyl group [35].In this study, we used Boc-glycine to graft pullulan polysaccharide to form a hydrophilic head, then combined it with cholesterol to form a hydrophobic tail by forming an ester bond to prepare the amphiphilic polymer, and then changed the amount of cholesterol to prepared NPs with different degrees of hydrophobic substitution. The size of different hydrophobic-substituted NPs differed, and particles with higher hydrophobic substitution were smaller. Thus, the self-assembly of the NPs was mainly driven by hydrophobic forces. Different hydrophobic substitutions of CHS/Pu-NH2 led to a slight difference in stability, but PDI showed that the stability of the three NPs was good in vitro.The particle size of the NPs increased after drug loading, and the higher the degree of substitution for CHS/Pu-NH2, the greater the drug loading. The higher drug loading did not reverse the difference in particle size caused by hydrophobic substitution, which also indicated that the size of NPs controlled by hydrophobic substitution was relatively stable. All particle types were positively charged, which is related to the positive electrical properties of amino groups. The zeta potential decreased slightly with increasing CHS/Pu-NH2 substitution and further decreased after the acid drug MTX was coated. Because of the positive electrical properties of the amino group, the corresponding amino ratio decreased with a large substitution of CHS/Pu-NH2 or increased proportion of amino groups.In vitro drug release studies showed that CHPN NPs could achieve the effect of slow release of drugs when used for drug delivery. With decreasing size, the drug sustained-release effect was significantly enhanced. This proves that the force affecting drug release was mainly hydrophobic rather than electrostatic. With the increase of hydrophobic force and the decrease of electrostatic attraction, drug release slowed down significantly.In the in vitro cytotoxicity study, drug release to matrix caused the cell cytotoxicity, and the positive potential of NPs could also play an important role in the absorption by tumor cells; it increased the cellular uptake, then release of drugs in cells, and thus higher cytotoxicity. CHPN1 NPs had the best effect in in vitro experiments, which had the lowest degree of hydrophobic substitution. The CHPN3 NPs with the highest degree of substitution had the lowest cell toxicity, which may be due to its poor release and NH2 modification. CHPN1/CHPN2 seemed to be more toxic than free MTX by higher uptake. The less proportion of NH2 results in a smaller positive NP surface, and high hydrophobic substitution results in poor flexibility, then caused a decrease of drug release and cellular uptake, so CHPN3 NPs cannot be so effective to tumor cells.The in vitro cytotoxicity study suggested that the hydrophobic substitution has a great influence on the function of NPs. Controlling the appropriate degree of hydrophobic substitution is of guiding significance for preparing NPs, inhibiting NPs on tumor cells, and improving the curative effect on tumor. The lower hydrophobic substitution NPs had the better inhibitory effect on cancer cells than higher hydrophobic substitution. This finding may be due to the fast release of NP drugs with low hydrophobic substitution, which enter cells in a free form to kill cells, and which is easy to be effectively absorbed by tumor cells [16, 36]. However, this experiment is still limited to the study at the cell level, and the in vivo behavior has not been studied, because the effectiveness of NPs also depends on their targeting and immune resistance. Targeting is largely related to size, and immune escape is related to size, surface properties, and deformation. In vivo efficacy also needs to consider the toxic side effects and also needs to release better NPs to reduce damage to normal cells. These characteristics are all affected by hydrophobic substitution, so the in vivo situation of NPs with different hydrophobic substitution degree needs further study.The study suggested the great effect of hydrophobic substitution on NP properties and function. It gives researchers a new instruction to prepare and control the size of NPs and gives us some inspiration to improve drug loading, optimize preparation methods, and improve cytotoxicity.


## 5. Conclusion

All prepared NPs have appropriate evenly distributed particle size, which are inversely proportional to hydrophobicity. With the increase of hydrophobic, the slow-release effect grows stronger, but the cancer cytotoxicity gets weaker. In this experiment, the hydrophobicity of the polymer has an important influence on particle size, drug loading, and drug release from NPs, and even cytotoxicity. The CHPN3 NPs with the highest degree of substitution had the smallest size, the highest drug loading, and the lowest cell toxicity. It provides a new mold for research to modify NPs in vitro and has a certain guiding significance for preparing appropriate size nanocarriers with an appropriate hydrophobic substitution degree.



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