Synthesis, protein delivery, and in vitro and in vivo toxicity of a biodegradable poly(aminoester)

Abstract

A hydrolytically degradable poly(amino ester) is synthesized and evaluated as a protein delivery vector in vitro. The poly(amino ester), with cleavable ester bonds, can degrade into nontoxic products both in vitro and in vivo under physiological conditions, exhibiting low toxicity. Our experiments reveal that the poly(amino ester) can efficiently condense proteins via electrostatic interaction, and is significantly less toxic than PEI 25 kDa. Using fluorescein isothiocyanate (FITC) labeled bovine serum albumin and ribonuclease A as model proteins, the degradable poly(amino ester) can efficiently deliver proteins into cells and regulate cell functions. These results suggest that the degradable poly(amino ester) is a promising and efficient protein delivery vector.

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Introduction

With the rapid progress in biotechnology, proteins have shown great promise in therapeutic applications by directing and regulating biological functions with few side-effects.¹ However, protein-based therapy poses significant challenges, including poor cellular internalization and loss of function.² In order to overcome these barriers, protein delivery systems such as hydrogels,^3,4 nanoparticles^5–8 and microspheres⁹ have received extensive attention. Although polymers have been developed as protein delivery vehicles,^10–12 there still remain some problems regarding formation of carrier–protein complexes via chemical coupling, including weakening of the protein activity and low loading efficiency.^10,13 Therefore, design and synthesis of novel polymer-based vectors with high cellular internalization, low toxicity, and biodegradation is highly desired.

Based on these factors, we developed a new strategy to form polymer/protein complexes via electrostatic interaction between positively charged polycations and negatively charged proteins. Cationic polymers are able to condense proteins and consequently facilitate cellular uptake. Among the current cationic vectors, polyethylenimine (PEI) is undoubtedly the most successful one. With increasing molecular weight, PEI exhibits improved transfection efficiency attributed to its high buffer capacity caused by the proton sponge effect,^14,15 but also an increase in cytotoxicity, which greatly limits its applications in vivo. Compared to the high molecular weight PEI, the low molecular weight PEI has much lower toxicity but almost no transfection activity.^16–22 In recent years, various types of hydrolytically or reductively degradable PEI polymers have been designed as delivery vectors.^16–22 These degradable PEIs have shown significantly enhanced transfection activity compared to the parent low molecular weight PEI and retain its biocompatibility. Considering the advantages of low molecular weight PEI, researchers focused on developing protein vectors based on low molecular weight PEI with high transfection efficiency as well as low cytotoxicity.

In order to improve the biodegradability and decrease the toxicity of polymer carriers, many researchers introduced degradable linkages, such as hydrolytically degradable ester, phosphoester, and disulfide bonds, to the polymer main chains or side chains.^23–27 Herein, we report the synthesis of a degradable PEI derivative via the Michael addition of PEI 800 Da to 1,4-butanediol diacrylate and its application as a polycationic carrier for protein delivery. Then the in vitro and in vivo toxicity and in vitro protein transfection efficiency of this polycation vehicle were also investigated.

Experimental section

Materials and instruments

Branched polyethylenimine (M_w = 25 kDa and 800 Da), 1,4-butanediol diacrylate, bovine serum albumin (BSA) and ribonuclease A (RNase A) were obtained from Sigma-Aldrich. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), OPTI-MEM and 0.25% Trypsin-EDTA were purchased from Gibco BRL (Grand Island, NY). Other chemicals of analytical grade were obtained from domestic suppliers and used as received.

Gel permeation chromatography (GPC) was carried out by using a Waters-2690D HPLC equipped with Ultrahydrogel 120, 250, and 2000 columns. Samples were detected with a Wyatt multi-angle light-scattering detector and a Waters 2410 differential refractive index detector. Zeta potential was measured by Nano-ZS (Malvern Instr.) at 25 °C. Confocal imaging was performed using a Nikon A1 laser confocal microscope.

Synthesis of degradable poly(amino ester)

The degradable poly(amino ester) (BDDA) was synthesized by Michael addition of PEI 800 Da to 1,4-butanediol diacrylate. In a two-neck flask, 4 g of PEI 800 Da was dissolved in 20 mL CH₃OH. The solution was bubbled with Ar for 30 min, then 1 g of 1,4-butanediol diacrylate was added, and the mixture was reacted at 50 °C for 24 h. The solution was then dialyzed against methyl alcohol (MWCO = 3500) for 5 days. The solvent was evaporated under reduced pressure, and 20 mL of cold water was added to dissolve the crude product. After lyophilization, the final product, polymer (BDDA) was obtained in 17% yield.

Polymer degradation study

The degradation of poly(amino ester) was performed according to the following typical procedure: 10 mg of BDDA was dissolved in 0.5 mL of phosphate buffer (0.1 M, dissolved in D₂O, pD 7.4) in an NMR tube, and the NMR tube was incubated at 37 °C. At certain intervals, the NMR tube was removed to measure ¹H NMR spectra.

Acid–base titration

The buffer capabilities of PEI 25 kDa, PEI 800 Da and BDDA were determined by an acid–base titration assay. 6 mg of each sample was dissolved in 30 mL of NaCl solution (150 mM). After titrating to pH 10 with 0.1 M sodium hydroxide, the solution was titrated to pH 3 with 0.1 M hydrochloride solution. The pH values were recorded with a pH meter.

In vitro toxicity assay

WST assay. The cytotoxicity of BDDA against in HeLa and HepG2 cells was evaluated by water soluble tetrazolium salts cell proliferation and cytotoxicity assay (WST assay), PEI 25 kDa, PEI 800 Da were used as the control. The WST assay is a colorimetric assay for measuring cellular metabolic activity, which reflects the number of viable cells. HeLa and HepG2 cell lines were separately seeded into 96-well plates at a density of 6000 cells per well in 100 μL of complete DMEM. When the cells were achieved 50–60% confluence after 24 h of incubation, 100 μL of polymer solution in DMEM at different concentrations was added to each well and incubated for further 24 h. Finally, the medium was replaced with fresh DMEM (100 μL), to which 10 μL of WST solution was then added and incubated for further 2 h. The absorbance at 450 nm was measured using a microplate reader (Perkin-Elmer Victor X4). The relative cell viability was calculated according to the following equation:

Relative cell viability (%) = 100 × (OD_sample − OD_background)/(OD_control − OD_background)

Reactive oxygen species measurement

The reactive oxygen species (ROS) level was monitored using a reactive oxygen species assay kit according to the protocol. Briefly, the cells were incubated with dichlorodihydrofluorescein diacetate (DCFH-DA) in medium for 30 min, and then washed three times. After treatment with different concentration of BDDA and PEI 25 kDa for 4 h, all samples were washed and the cells were suspended in 1 mL PBS. Flow cytometry analysis (FACS) was performed immediately with the excitation/emission wavelength at 488/530 nm.

In vivo toxicity assay

Six-week-old male Balb/c mice (Suzhou Belda Bio-Pharmaceutical Co.) were raised in an animal facility under filtered air (16–22 °C) and fed with standard pellet diet and pure water. The study was performed in accordance with the Guidelines for the Care and Use of Research Animals. For the BDDA in vivo toxicity experiments, 20 rats were equally and randomly divided into 4 groups, that five mice per group were subjected to the analysis. The mice were tail intravenously injected 200 μL of 0.9% NaCl as the control group, and 200 μL of BDDA solution at concentrations of 1.0 mg mL⁻¹, 1.5 mg mL⁻¹ and 3.0 mg mL⁻¹ (corresponding to doses of 10 mg kg⁻¹, 15 mg kg⁻¹ and 30 mg kg⁻¹), respectively. Finally, the mouse body weights were measured at several time points. In the 15^th day, the mice were sacrificed to collect blood for hematology analysis, and organs, including heart, liver, spleen, lungs and kidneys for histology analysis.

Morphology characterization

Atomic force microscopy (AFM) images were taken with a Veeco Dimension 3100 atom force microscope. Transmission electron microscopy (TEM) images were obtained on a Tecnai G2 F20 S-Twin transmission electron microscope. The samples for morphological analyses were freshly prepared with 1 mg mL⁻¹ BDDA and 20 mg mL⁻¹ BSA. TEM samples were then stained with 2% tungstophosphoric acid.

Cellular uptake of polycation/protein complexes

For the protein delivery experiment, HeLa cells were seeded into a 24-well plate and incubated to 70% confluence before transfection with 900 μL media. Firstly, all of the polycation/protein complexes were prepared fresh. Appropriate amounts of polymer solution (in NaHCO₃/Na₂CO₃ buffer) were added to the protein solution to achieve desired w/w ratios, and the complexes were mixed by vortexing for 5 s and incubated for 30 min at room temperature. Then, 100 μL of polycation/protein complexes containing 1 μg or 2 μg of BDDA with different concentrations of protein were added. The cells were incubated with BDDA/BSA-FITC for a further 4 h, and then rinsed with PBS 3 times. Finally, confocal fluorescence microscopy and flow cytometry were used to investigate cellular uptake of BDDA/BSA-FITC. Free BSA-FITC with same concentration was also incubated with cells as a control. For the induced apoptosis/necrosis by BDDA transfected RNase A experiment, BDDA and RNase A complexes at different concentrations were treated with cells for 65 h, and then the percentage of dead cells was examined by WST assay.

Result and discussion

Synthesis and characterization of polymer

The aim of the study is to develop a novel protein vector based on degradable poly(amino ester). The polymer was synthesized by Michael polyaddition of branched PEI 800 Da to 1,4-butanediol diacrylate (Scheme 1). It is expected that the presence of different types of amino groups in the cationic vector could efficiently improve protein delivery efficiency. The primary amines the polymer bears are capable of condensing proteins into polycation/protein complexes through electrostatic interaction, while the secondary and tertiary amines provide the polycation with high buffer capacity, which can facilitate the endosomal escape of polycation/protein complexes.

Scheme 1 The synthetic route to BDDA.

The polymer were purified by dialysis against anhydrous methyl alcohol (MWCO = 3500) and lyophilized to obtain BDDA, and the weight-average molecular weight (M_w) of BDDA was detected by GPC, M_w = 9.8 × 10³, PDI = 2.24.

The degradation of the polymer was confirmed by ¹H NMR at 37 °C in phosphate buffer (D₂O, 0.1 M, pD 7.4). Fig. 1 shows ¹H NMR spectra of BDDA after 0, 5 and 24 h degradation in phosphate buffer. The extent of degradation was determined by the integral of signal at 3.6, which was attributed to the CH₂ protons adjacent to the hydroxyl group (HOCH₂). The result also illustrated that BDDA is readily degraded with a faster rate at pH 7.4, at which the degradation of ester bonds was observed within 5 h. Similar results have also been reported by other groups.^28,29

Fig. 1 (A) Diagram showing BDDA degradation. (B) ¹H NMR spectra of BDDA degraded in deuterated phosphate buffer (0.1 M, pH 7.4) with time.

Buffer capacity

According to the proton sponge hypothesis,^21,22 cationic polymers with high buffer capacity in the range pH 5–7, such as PEI, may play an important role in endosomal escape, and consequently lead to high transfection efficiency. In this study, the buffer capacities of BDDA, PEI 25 kDa and 800 Da were evaluated by acid–base titration in 150 mM NaCl solution. As shown in Fig. 2, the buffer capacity of PEI 25 kDa and PEI 800 Da is similar, which is slightly higher than that of BDDA. Previous work has demonstrated that the buffer capacity of polycation mainly depends on the presence of primary, secondary and tertiary amine groups. The density of amines in BDDA decreased due to the introduction of 1,4-butanediol diacrylate, thus causing loss of buffer capacity. This may explain why the buffer capacity of BDDA is lower than that of PEI.

Fig. 2 Acid–base titration curves of PEI 25 kDa, PEI 800 Da and BDDA in 150 mM NaCl solution.

Toxicity assessment

Cell viability assay. Biocompatibility of a polymer is very important for its application in protein delivery. The relative cellular viability of BDDA, PEI 25 kDa and PEI 800 Da was evaluated in HeLa and HepG2 cells. As shown in Fig. 3, PEI 800 Da showed very low cytotoxicity in both HeLa and HepG2 cell lines. The cytotoxicity of BDDA and PEI 25 kDa was found to be concentration dependent, as the cell viability decreased with increasing polymer concentration. In comparison, PEI 25 kDa showed the highest toxicity both in HeLa and HepG2 cell lines, only 20% and 40% of cells survival at 10 μg mL⁻¹, while BDDA exhibited 70% and 90% of cells survival at 10 μg mL⁻¹ in HeLa and HepG2 cell lines, respectively. The in vitro cytotoxicity experiment indicated that BDDA is a more biocompatible carrier than PEI 25 kDa.

Fig. 3 Cell viability of BDDA incubated with (A) HeLa and (B) HepG2 cells determined by WST assay. PEI 25 kDa and PEI 800 Da were used as control.

ROS level

Oxidative stress is implicated as a mechanism of cytotoxicity.^30,31 We explored the generation of reactive oxygen species (ROS) to determine the cytotoxicity caused by oxidative stress of BDDA. As shown in Fig. 4, the generation of ROS is polymer concentration dependent. After 4 h of BDDA treatment, the level of ROS induced by 20 μg mL⁻¹ of BDDA was only 1.3-fold compared to the control group, while the generated ROS level was significantly increased for the same concentration of PEI after 4 h exposure (2.5-fold compared to the control group). Clearly, compared to PEI (25 kDa), BDDA did not significantly increase ROS level, and BDDA at lower concentration (<5 μg mL⁻¹) did not influence the generation of ROS.

Fig. 4 Effect of BDDA and PEI (25 kDa) on ROS generation.

In vivo toxicity of BDDA

In our experiment, the in vivo toxicity of BDDA at different dosages (10 mg kg⁻¹, 15 mg kg⁻¹ and 30 mg kg⁻¹) was investigated after injection at the 1^st, 4^th, 7^th and 10^th day using male Balb/c mice.

Initially, we investigated the basic physical signs of toxicity of BDDA. First of all, the effect of BDDA on the body weight of the mice was examined. Throughout the experiment, mice intraperitoneally injected with 10 mg kg⁻¹, 15 mg kg⁻¹ and 30 mg kg⁻¹ BDDA 4 times did not show unusual responses or behaviors, such as lethargy in two weeks. In addition, no significant weight loss compared to the control group was observed. As shown in Fig. 5(A), the body weights of the control group and the BDDA injection groups (dosage of 10 mg kg⁻¹ and 15 mg kg⁻¹) maintained similar increasing trend over two weeks. Only the highest dose group exhibited a slight decrease of weight, probably due to accumulation of BDDA in the body. These results suggested that the injected BDDA did not perceivably interfere with the growth of mice.

Fig. 5 (A) Body weight changes of the male Balb/c mice treated with BDDA at the dose of 10 mg kg⁻¹, 15 mg kg⁻¹ and 30 mg kg⁻¹ at different time points. (B) Images and (C) weight ratio of these organs (mg per 100 g) from BDDA treatment groups and control group.

To further study the in vivo toxicity of BDDA, histological assessment of tissues was conducted to determine whether the polymer itself or its degradation products cause tissue damage, inflammation, or lesions from toxic exposure. The tissue morphology (Fig. 5(B)) and the weight ratio of organs (mg per 100 g), including liver, spleen, kidney, lung and heart were measured to explore the tissue toxicity (as shown in Fig. 5(C)). No significant changes were revealed from three treated animal groups and the control group. Representative histology results are shown in Fig. 6. Hepatocytes in the liver samples were observed to be normal, and no signs of inflammatory response were observed. No pulmonary fibrosis was detected in the lung samples. Necrosis was not found in any of the samples. Generally, there were no apparent pathological changes or lesions related to treatment of the animals with BDDA in histological analysis of the heart, lung, kidney, liver and spleen.

Fig. 6 Histological images of the major organs of mice after injection of different doses of BDDA. Tissues were collected from heart, kidney, liver, lung, and spleen, and then stained with standard haematoxylin and eosin (scale bar = 200 μm).

Moreover, administration of BDDA to the mice may cause changes in hematology indicators such as red blood cells or hemoglobin due to toxicity. We chose the following standard hematology markers for hematology evaluation: red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelet count (PLT), white blood cell count (WBC), neutrophil cell count (NEU), lymphocyte count absolute value (LYM), mononuclear cell count (MONO), eosinophil cell count (EOS), and basophilic granulocyte count (BASO). Representative hematology results presented in Fig. 7 indicate that all the measured factors are within normal ranges and do not indicate a trend associated with treatment. There were no significant differences in hematology measurements for BDDA injection with 10 mg kg⁻¹, 15 mg kg⁻¹ and 30 mg kg⁻¹.

Fig. 7 Hematology results of the mice treated with BDDA at different dosages. These results show mean and standard deviation of (a) RBC, (b) HGB, (c) HCT, (d) MCV, (e) MCH, (f) MCHC, (g) RDW, (h) PLT, (i) WBC, (j) NEU, (k) LYM, (l) MONO, (m) EOS and (n) BASO.

In summary, mice injected with 10 mg kg⁻¹, 15 mg kg⁻¹ and 30 mg kg⁻¹ of BDDA stay healthy, without any evidence of toxicity. All measured biochemical markers were in the normal range. All toxicity studies in Balb/c mice show that BDDA with specific concentrations (10 mg kg⁻¹, 15 mg kg⁻¹ and 30 mg kg⁻¹) has very low in vivo toxicity. A possible reason is that BDDA may be degraded into smaller molecules in the body, which would reduce the toxicity of the cationic polymer vector.

Characterization of polycation/protein complexes

We first employed BSA as a protein model to study the morphology of BDDA/protein complexes by TEM and AFM. As presented in Fig. 8, BDDA/BSA formed randomly shaped nano-sized particles with spherical, rod and oval-like morphologies, with thickness of about 5 nm (Fig. 8(B)), suggesting successful formation of polycation/protein complexes mainly via electrostatic interaction. As positive surface charge of polymer/protein polyelectrolyte complexes is another important factor for cellular uptake and efficient transfection. The zeta potential of BDDA/protein complexes was then measured to assess their surface charge. The surface charge of BDDA/BSA was 21.03 mV, while the zeta potentials of BDDA and BSA were 37.49 mV and −11.37 mV, respectively. The results of zeta potential measurement suggested successful formation of positive BDDA/protein complexes, which facilitated penetrating the negative charge cellular membranes and then enhanced cellular uptake.

Fig. 8 (A) TEM (scale bar = 50 nm), and (B) AFM images of BDDA/BSA complexes.

Cellular uptake of polycation/protein complexes

Similarly to gene delivery, cellular uptake of polymer/protein complex is necessary for efficient protein therapy.³² Like genes, proteins can be condensed with cationic polymers via electrostatic interactions. It is expected that the positively charged BDDA/protein complexes may facilitate binding with the negative charged cell membrane, which initiates cellular internalization.^33,34 In addition, BDDA could be substantially degraded under physiological conditions, which could help the release of proteins from the complexes. In the present study, FITC labelled BSA delivered by BDDA was measured and tracked. Its transfection efficiency was evaluated by confocal fluorescence microscopy and flow cytometry.

Cells treated with BDDA at different concentrations condensed with 40 μg mL⁻¹ BSA-FITC showed green fluorescence localized in cytoplasm, while no fluorescence was observed for free BSA-FITC under the same experimental conditions (Fig. 9(A)). The nucleus stained with DAPI appears colored blue in dark field. The cellular internalization efficiency of BSA-FITC via BDDA was significantly higher than that of free BSA-FITC, suggesting the indispensable role of BDDA in transporting proteins into the cells. In addition, these images also indicate the cellular internalization of the different w/w ratio of BDDA for protein. With increasing the concentration of BSA-FITC (Fig. 9(A) c and d), the amount of BSA-FITC transported into cells also increased. As a result, more green fluorescence of BSA-FITC was observed at cytoplasm, indicating the capability of BDDA to bind and deliver proteins to cells.

Fig. 9 (A) Confocal fluorescence microscopy images of HeLa cells (a) before and after incubation with (b) BSA-FITC, (c) BDDA (1 μg mL⁻¹)/BSA-FITC (5 μg mL⁻¹), and (d) BDDA (1 μg mL⁻¹)/BSA-FITC (10 μg mL⁻¹). (B) Representative flow cytometry histograms of cellular uptake of BDDA/BSA-FITC.

Moreover, to further confirm the result from the fluorescence images above, flow cytometry was performed. The mean fluorescence intensity values (Fig. 9(B)) also reveal that the amount of BSA-FITC transported by 1 μg mL⁻¹ BDDA to cytoplasm is much higher than that of free BSA-FITC (40 μg mL⁻¹). BDDA (1 μg mL⁻¹)/BSA-FITC (40 μg mL⁻¹) exhibited strong fluorescence, being nearly 200-fold higher than that of free BSA-FITC in HeLa cells. Furthermore, the protein delivery efficiency increased significantly when the concentration of BDDA increased for the BDDA/protein complexes, due to more effective protein complexation.

Induced apoptosis by BDDA transfected RNase A

In order to understand the regulation of cell functions by the proteins transfected via BDDA, we checked the cytotoxicity of BDDA/RNase A complexes using HeLa cells. RNase A, a cleaving protein enzyme, was used in our experiment. RNase A has been proven to degrade mRNA as well as tRNA nonspecifically, to inhibit protein synthesis in cytoplasm, and to modulate cell apoptosis.^35,36 WST results reveal that BDDA induced a significant increase of cell death. As shown in Fig. 10(A), nearly 10% to 30% increasing of induced cell death was observed after being treated with BDDA (2 μg mL⁻¹) combination with RNase A of different concentration (20 μg mL⁻¹, 40 μg mL⁻¹ and 70 μg mL⁻¹), above the basal cytotoxicity caused by either BDDA or RNase A alone. With increasing the protein concentration in the BDDA/protein complexes, the apoptosis/necrosis induced by RNase A also increased, as more proteins were transported into cells efficiently.

Fig. 10 Relative viability of HeLa cells incubated with (A) RNase A (70 μg mL⁻¹), BDDA (2 μg mL⁻¹)/RNase A (20 μg mL⁻¹), BDDA (2 μg mL⁻¹)/RNase A (40 μg mL⁻¹), BDDA (2 μg mL⁻¹)/RNase A (70 μg mL⁻¹), and BDDA (2 μg mL⁻¹), respectively. (B) BDDA at different concentrations (1 μg mL⁻¹, 1.5 μg mL⁻¹ and 4 μg mL⁻¹) with or without condensing 70 μg mL⁻¹ RNase A.

To further investigate the protein delivery efficiency, the transfection of RNase A (70 μg mL⁻¹) by BDDA with different concentrations was explored. BDDA at different concentrations (1 μg mL⁻¹, 1.5 μg mL⁻¹ and 4 μg mL⁻¹) condensed with RNase A induced similar level of cell death (Fig. 10(B)). It might be possible that BDDA at a concentration of 1 μg mL⁻¹ has already condensed with most of the proteins, and consequently delivers the proteins efficiently and induces cell apoptosis/necrosis. These results suggest the feasibility of BDDA as a promising bioactivity protein delivery vector for protein transfection and cell function regulation.

Conclusions

In summary, a hydrolytically degradable poly(amino ester) was synthesized and explored for delivery of proteins into cytoplasm. Such a vector, we demonstrated, showed high transfection efficiency in vitro, good biocompatibility and low toxicity both in vitro and in vivo. Utilizing biodegradable cationic polymer to deliver functional proteins into cytoplasm and subsequently regulate cell metabolism might open an ideal process of protein transfection. These results suggested that the degradable poly(amino ester) is a promising candidate for protein delivery with high efficiency and low toxicity.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 51361130033 and 21073224), and Innovation Project of CAS (KJCX2.YW.M12).

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Footnote

† These authors contributed equally to this work.

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