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Wei Wei, Guanghui Ma et al. Macrophage responses to the physical burden of cell-sized particles

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Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x

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Fabrication of aminated poly(glycidyl methacrylate)sbased polymers for co-delivery of anticancer drug and p53 gene Lei Lia,b, Hongrui Tiana, Jinlin Hea, Mingzu Zhanga, Zuguang Lib, Peihong Nia,* Aminated poly(glycidyl methacrylate)s-based polymers as gene delivery not only can reduce toxicity and improve solubility, but can improve gene transfection efficiency and reduce protein aggregation. In this study, we first prepared poly(glycidyl methacrylate) (PGMA) via reversible addition-fragmentation chain transfer (RAFT) polymerization, and then the produced PGMA was post-modified with ethanol amine (EA), 1-amino-2-propanol (AP), 3-(dibutylamino)propylamine (DA) and N-(2-hydroxyethyl)ethylenediamine (HA), respectively, to yield four kinds of PGMA-based gene vectors containing hydroxyl groups (abbreviated as PGEA, PGAP, PGDA and PGHA). The effects of the different side chains and hydroxyl groups on the biological properties of these four cationic polymers were investigated. We found that the transfection efficiency of PGHA/p53 complex was higher than those of the other three polymer/gene complexes through MTT assay and laser scanning confocal microscope. Hence, we chose HA for further post-modification to fabricate a cationic copolymer, PCL-ss-P(PEGMA-co-GMA) (abbreviated as PGHAP), via a combination of ring opening polymerization (ROP) and RAFT copolymerization. The PCL-ss-P(PEGMA-co-GMA) amphiphilic copolymer could self-assemble into nanoparticles, which could be used to encapsulate anticancer drug doxorubicin (DOX) and compress p53 gene to form DOX-loaded PCL-ss-P(PEGMA-co-GHA)/p53 complex (abbreviated as DPGHAP/p53). The gel retardation assay showed that p53 gene could be well immobilized and maintained stably under the electronegative conditions. MTT assay showed that DPGHAP/p53 complex had a significant antitumor effect on A549 cells and H1299 cells compared with free DOX or/and p53 gene therapy alone. Furthermore, the test results from live cell imaging system revealed that the DPGHAP/p53 complexes could deliver effectively DOX and p53 gene into A549 cells. Therefore, the constructed cationic polymer PCL-ssP(PEGMA-co-GMA) has potential application prospects as a co-vector of anticancer drugs and genes.

Introduction Cancer is considered the main cause of death and the biggest barrier to increasing life expectancy in the 21st century.1, 2 The annual incidence and mortality rate of lung cancer rank second in the world according to the global cancer database 2018

aCollege

of Chemistry, Chemical Engineering and Materials Science, State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Suzhou Key Laboratory of Macromolecular Design and Precision Synthesis, Soochow University, Suzhou 215123, P. R. China. Tel: +86-512-65882047; *E-mail: [email protected] bCollege of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China †Electronic supplementary information (ESI) available: Experimental details and characterization data. 1H NMR spectra of CPDB, CPDB-ss-OH, PCL40-ss-CPDB. GPC traces of PGMA, PCL40-ss-CPDB and PCL60-ss-CPDB, and PCL40-ss-P(PEGMA7-co-GMA80). The pictures of polymeric solubility of PGDA, PGEA, PGAP, and PGHA homopolymers. FT-IR spectra of PGEA, PGAP, and PGHA. Zeta potential of PGEA, PGAP, and PGHA aqueous solution. Zeta potential of PGEA/DNA, PGAP/DNA, PGHA/DNA, and PEI/DNA complexes. Agarose gel electrophoreses of PGEA/DNA, PGAP/DNA, PGHA/DNA, and PEI/DNA complexes. Cell viabilities of A549 cells and H1299 cells treated with free p53, PGEA/p53, PGAP/p53, and PGHA/p53 complexes. XPS spectra of PCL40-ss-P(PEGMA7-coGMA80). See DOI: 10.1039/xxxx

(GLOBOCAN 2018 database).2, 3, 4 Chemotherapy has been widely investigated for the treatment of numerous cancers. However, the conventional chemotherapy based on small-molecular drugs remains less successful mostly due to the undesired side effects, low therapeutic efficiency as well as drug resistance.5, 6 To solve these issues, as a new approach for the treatment of lung cancer, the combination therapy using different therapeutic agents has attracted more attention, especially co-delivery of anticancer drugs and genes.7-9 Since the mutation and dysfunction of protein 53 (p53) has been associated with over 50% of human cancers, it is clear that p53 has great significance in cancer growth and therapy.10, 11 The major challenge is to find appropriate vectors to immobilize genes efficiently. Recently, non-viral gene vectors have received more attention due to their low host immunogenicity, ease of preparation, and relative safety. For example, polyethylenimine (PEI)12 and poly(2-dimethylamino)ethyl methacrylate (PDMAEMA)13 have been used as pDNA carriers. However, most of these nanocarriers have drawbacks such as a high toxicity and poor biocompatibility, which gravely limited their potential applications in clinical trials.14 The key problem is how to construct a cationic polymer delivery system, which can not only improve the transfection

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efficiency of genes, but also reduce the cytotoxicity of the polymer.15, 16 Shielding extra charges of complexes, which can impair the effect between polycations and components in cell, is one powerful method to decrease the cytotoxicity of gene vectors.17, 18 The epoxide groups of PGMA can react irreversibly with nucleophilic groups, such as –NH2, and yield many hydroxyl groups on the side groups.19, 20 Such aminated PGMA was reported as a safe gene vector.19, 21, 22 It is well known that a wide range of new hydroxyl-rich polycationic vectors have been applied for effective nucleic acid delivery. In addition, sufficient hydroxyl groups provide hydrophilicity and blood compatibility, which are beneficial for transfection performances.23-25 In order to study the effect of the side hydroxyl groups of polymers on the performance of gene carriers, Li et al. prepared four kinds of cationic polymers based on PGMA, (abbreviated as PAEMA, PAHPMA, PAEAEMA and PAEAHPMA, respectively), with similar chemical structures and degrees of polymerization via atom transfer radical polymerization (ATRP). They found that the numbers of secondary amine and hydroxyl groups have a great effect on cytotoxicity and transfection efficiency of gene vectors.26 The hydroxyl groups might increase the bonding capacity to DNA and at the same time decrease the surface charge of the polymer/DNA complexes due to the formation of hydrogen bonds between the polymers and DNA.26-28 Hence, it is important for gene vectors to choose the number of secondary amine and hydroxyl groups. In addition, as a typical biomaterial approved by the United States Food and Drug Administration (FDA), poly(ε-caprolactone) (PCL) have been widely used to construct amphiphilic copolymers, which can encapsulate hydrophobic drug doxorubicin (DOX), camptothecin (CPT), and/or paclitaxel (PTX) as drug delivery.29, 30 PEGylated complexes can exhibit poor stability and premature unpackaged in vivo, possibly because of interactions with serum proteins and extracellular matrix components.31, 32 Environmentally sensitive polymers or stimulus-responsive bonding groups can provide specific properties to drug or gene carriers. In addition, the cytosolic concentration of glutathione (GSH) is three orders of magnitude higher than the extracellular concentration, which will cause the disulfide bonds (S-S) of vector to be cleaved and promote drug and/or gene delivery.33, 34 In recent years, with the development of controlled/living polymerization such as ATRP,35 RAFT,36, 37 and nitroxide-mediated polymerization (NMP), most of methacrylic monomers can be polymerized to yield sophisticated (co)polymers, which can be further transformed into functional materials with different structures and applications.38-40 Pun and co-workers reported a pHsensitive block-statistical copolymer PCL-SS-P-{(GMA-TEPA)-stOEGMA}, which could provide a solution to stability and high transfection efficiency dilemma.41 Wu and co-workers prepared a series of acid-labile -CD-PGEA vectors via incorporating the acetal bridges into star-shaped PGMA derivatives, which could condense pDNA into the compact complexes with high cellular uptake and provide preconditions for high transfection efficiency.42 Recently, our group reported a kind of composite nanoparticles composed of a pH-sensitive prodrug mPEG-b-PBYP-hyd-DOX and a polycation genetic vector mPEG-b-PBYP-g-DAE. The hybrid micelles could codeliver the tumor-suppressor p53 gene and anticancer drug DOX into A549 cells by endocytosis. This is a promising approach for the

treatment of lung cancer with a combination of therapy DOXOnline and Viewof Article DOI: 10.1039/D0TB01811B p53 gene.43 Moreover, the precise synthesis and regulation of polymeric structures, including charge density, will play a better role in improving the performance of drug and gene common carriers. In this regard, a combination of ROP and RAFT copolymerization can be used to obtain a series of carriers with new structures, depending on the monomer structure. Herein, we first prepared poly(glycidyl methacrylate) (PGMA) via RAFT polymerization, which can be post-modified through ethanol amine (EA), 1-amino-2-propanol (AP), 3(dibutylamino)propylamine (DA) and N-(2hydroxyethyl)ethylenediamine (HA), respectively, as shown in Scheme 1. The performance of the polycations as gene vectors were evaluated through transfection efficiency and cytotoxicity. Then, PCL-ss-P(PEGMA-co-GMA) was prepared by a combination of ROP and RAFT copolymerization, the epoxide groups of PCL-ssP(PEGMA-co-GMA) pendants were post-modified using HA to yield the PCL-ss-P(PEGMA-co-GHA) polycation as a co-vector of DOX and p53 gene (Scheme 2). The polycaition could self-assemble into coreshell nanoparticle in aqueous solution, which could not only encapsulate DOX and immobilize p53 gene due to the presence of hydrophobic PCL segments and positive charge shell, but also maintain the stability of the complex. Once the complex nanoparticles were internalized into tumor cells, the rich hydroxyl groups in the complex will facilitate transmembrane transport of the nanoparticles and escape from the endosome. In the reductive medium, when the disulfide bond in the complex nanoparticles were cleaved, the encapsulated DOX and p53 gene could be released to inhibit the proliferation of tumor cell (Scheme 3). Hence, we provide a convenient method for fabricating the redox responsive polycation as co-carrier of DOX and p53 gene, which could improve the gene transfection efficiency and overcome the multidrug resistance effectively in tumor cells.

Scheme 1. Synthetic route of PGEA, PGAP, PGAP and PGHA cationic gene delivery. Among them, R1, R2, R3 and R4 represent ethanol amine (EA), 1-amino-2-propanol (AP), 3-(dibutylamino)propylamine (DA), and N-(2-hydroxyethyl)ethylenediamine (HA), respectively.

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Scheme 2. Synthetic route of PCL-ss-P(PEGMA-co-GHA) copolymer.

Scheme 3. Schematic illustration of the self-assembly of DPGHAP/p53-GFP complex, which could form the nanoparticles and would be disrupted under an intracellular acidic and GSH microenvironment of tumor cells and further result in gene and drug co-delivery.

Results and Discussion Characterization of PGMA-based Homopolymer. Firstly, we prepared poly(glycidyl methacrylate) (PGMA) via RAFT polymerization. Four kinds of modified PGMA (abbreviated as PGEA, PGAP, PGDA and PGHA, respectively) with flanking cationic secondary amine and nonionic hydrophilic hydroxyl groups were readily prepared via ring-

opening reaction of the pendant epoxide groups of PGMA with the amino moieties of EA, AP, DA and HA, respectively. Then, PCL-ss-P(PEGMA-co-GHA) copolymer was prepared by a combination of ROP and RAFT copolymerization. PGHAP was obtained through a post-modification of PGMA with HA. The chemical structures, molecular weights, and element contents of above-mentioned polymers were characterized.


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The typical structure of PGMA was characterized by 1H NMR spectroscopy as shown in Figure 1, and Figure S1 for CPDB in the Supporting Information. For PGMA (Figure 1), the signals at δ 3.80 ppm and δ 4.32 ppm correspond to the methylene protons adjacent to the oxygen moieties of the ester linkages (a, −CH2−O−C=O). The peaks (b and c) at δ 3.24 ppm, δ 2.64 ppm and δ 2.85 ppm can be assigned to the protons of the epoxide group.

Figure 1. 1H NMR spectrum of PGMA in CDCl3.

1.13 and the M̅n value of PGMA homopolymer was 7.4103 g/mol. Figure S3 showed the solubility of these four kinds of polymers in water, from which we could see that all three samples are good water solubility except for PGDA. So the structure of PGDA polymer was not further characterized because of its poor water solubility. The chemical structures and element contents of PGEA, PGAP, and PGHA were characterized by 1H NMR, FTIR, and XPS analysis, respectively, as shown in Figure 2, Figure S4, and Figure 3. From Figure 2(A, B and C), we can find that the chemical shifts are all attributed to the protons of PGEA, PGAP, and PGHA. The detailed information was provided in the Supporting Information. The stretching vibration peaks of C=O and C=N in PGEA, PGAP and PGHA appeared in 1727 cm-1 and 1150 cm-1, respectively. (Figure S4) The results of XPS analysis showed that C1s, N1s, and O1s core-level spectra of PGEA, PGAP, and PGHA could be curvefitted into different unimodal components in Figure 3. For example, the detailed information of PEGA is given as follows: Binding energies (BEs) at 284.8 eV, 286.2 eV, and 289.0 eV attributable to the C−C, C−N−C, and O=C−O species, BEs at 401.4 eV and 399.4 eV attributable to N-H and C-N species, with BEs at 532.7 eV and 532.0 eV attributable to the O-H and C=O species, respectively.22, 44, 45 Moreover, the element contents of C, N, and O in PGEA, PGAP, and PGHA were calculated by XPS analysis. The detailed information was listed in Table S1.

Figure 3. XPS spectra of PGEA, PGAP, and PGHA: (a) Survey scan, (b) C 1s, (c) N 1s, and (d) O 1s.

Cell Viabilities of PGEA, PGAP, and PGHA Polymers. Figure 2. 1H NMR spectra of (A) PGEA, (B) PGAP, and (C) PGHA in D2O. The GPC trace of PGMA homopolymer in Figure S2 exhibited a unimodal peak with a polydispersity index (Ð) of

By MTT assay, Figure 4(A, B, and C) showed that both PGEA, PGAP, and PGHA maintained relatively high cell viabilities against all cell lines (L929 cells, A549 cells, and H1299 cells) within a certain polymer concentration range. In contrast, the cytotoxicity curves of PEI (10 kDa) presented sharply drops during the whole concentration range. This means that the

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C). The zeta potential values increased from negative value to positive value with the increase of N/P ratio.


Figure 5. Agarose gel electrophoreses of (a) PGEA/DNA, (b) PGAP/DNA, (c) PGHA/DNA, and (d) PEI/DNA complexes at different N/P ratios. (Lane 1 is DNA control, lane 2-8 correspond to N/P ratios of 0.5, 1, 2, 3, 4, and 5, respectively) The ability of cationic polymers to bind DNA was also examined by agarose gel electrophoresis. Figure 5(a, b, c and d) shows the gel retardation results of PGEA/DNA, PGAP/DNA, and PGHA/DNA complexes with increasing N/P ratios in comparison with that of linear PEI (10 kDa). The PGEA/DNA, PGAP/DNA, and PGHA/DNA complexes could condense plasmid DNA completely when N/P ratio was 1.0. Furthermore, for verifying the stability of the PGEA/DNA, PGAP/DNA, and PGHA/DNA complexes, we used heparin sodium to simulate the in vivo environment. The results indicated that the PGEA/DNA, PGAP/DNA, and PGHA/DNA complexes had good stability in comparison with that of PEI/DNA complex when adding into an appropriate amount of heparin sodium (Figure S7).

Figure 4. Cell viabilities of PGEA, PGAP, PGHA, and PEI polymeric solution at different concentrations against L929 cells (A), A549 cells (B), and H1299 cells (C) for 48 h of incubation.

Gel Retardation Test and Gene Transfection Assay It is crucial factors about their zeta potential and biocompatibility for the polymers as gene carriers. As shown in Figure S5, the zeta potential values of PGEA, PGAP, and PGHA solution were +23.4 mV, +28.2 mV, and +42.2 mV, respectively. Subsequently, the zeta potentials of PGEA/DNA, PGAP/DNA, PGHA/DNA and PEI/DNA complexes were also measured by DLS measurement. The zeta potentials of PGEA/DNA, PGAP/DNA, and PGHA/DNA were about +20 mV in the range of the nitrogen/phosphate (N/P) ratio  1.0 in Figure S6(A, B and

Figure 6. Representative images of expression of A549 cells as examined after 48 h of transfection with (A) Free p53-GFP, (B) PEI/p53-GFP (C) PGEA/p53-GFP, (D) PGAP/p53-GFP, and (E) PGHA/p53-GFP, (N/P=3) using confocal laser scanning microscope. (The concentration of p53-GFP is 6 mg/L. Scale bar=50 μm) Using the green fluorescent protein-labeled p53 gene (p53-GFP), we determined the in vitro gene transfection efficiency of the cationic polymers/p53-GFP complexes by confocal laser scanning microscope. Figure 6 shows gene transfection efficiency of PGEA/p53-GFP, PGAP/p53-GFP, and PGHA/p53-GFP in comparison with those of Free p53 gene and linear PEI (10 kDa)/p53-GFP against A549 cell. The results confirmed that these three polymers/p53-GFP complexes possess strong green fluorescence, indicating that all the three samples have good transfection efficiency.


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flanking hydroxyl groups of polycation probably well shielded the deleterious cationic charges of gene carriers.19



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For calculating the fluorescence intensity of PGEA/p53GFP, PGAP/p53-GFP, and PGHA/p53-GFP quantitatively, the green fluorescence of these complexes were determined by confocal laser scanning. Figure 7 shows the typical images of p53-GFP expression mediated by different polycation/p53-GFP complexes against A549 cell at the N/P ratio of 3 (the concentration of p53-GFP was 6 mg/L). The fluorescence intensity of PGEA/p53-GFP, PGAP/p53-GFP, and PGHA/p53-GFP transfection systems were stronger than that of free p53-GFP as control, in which the signal of PGHA/p53-GFP had the highest fluorescence intensity.

The specific synthesis method of the common vector is as follows: CPDB-ss-OH was first prepared via esterification reaction. Then, PCL-ss-P(PEGMA-co-GMA) copolymer were prepared by a combination of ROP and RAFT copolymerization. Finally, PCL-ss-P(PEGMA-co-GHA) polycation was obtained by post-modification using small molecule HA (Scheme 2). Figure S9 and Figure S10 show the 1H NMR spectra of CPDB-ss-OH and PCL-ss-CPDB, respectively. In addition, Figure S11, and Figure S12 show GPC traces of PCL-ss-CPDB and PCL40ss-P(PEGMA7-co-GMA80) copolymers. As shown Figure 8, the signals at δ 3.82 ppm and δ 4.31 ppm (c, −CH2−O−C=O) corresponded to the methylene protons adjacent to the oxygen moieties of the ester linkage. The peaks at δ 3.24 ppm, δ 2.64 ppm and 2.85 ppm were assigned to the methylidyne (peak d, −CH2−CH(O)−CH2) and (peak e, CH−CH(O)−CH2) methylene protons of the epoxide group, respectively. The chemical shifts at δ 4.06 ppm (peak c’), δ 3.65 ppm (peak d), δ 4.06 ppm (peak c’), δ 3.38 ppm (peak e’), δ 4.06 ppm (peak i’), δ 2.31 ppm (peak r), δ 1.65 ppm (peak g), and δ 1.38 ppm (peak h) were ascribed to PEGMA and PCL segments of PCL-ss-P(PEGMA-co-GMA), respectively. The polymerization degree of PCL-ss-CPDB and PCL-ss-P(PEGMA-coGMA) were calculated according to the 1H NMR analysis by the eqs (S1), (S2) and (S3) in the Supporting Information.

Figure 7. Quantitative determination of GFP fluorescence intensity within A549 cells that incubated 48 h with free p53GFP, PGEA/p53-GFP, PGAP/p53-GFP, and PGHA/p53-GFP at selected N/P ratio (N/P=3) by confocal laser scanning. In other test, A549 cells and H1299 cells were cultured in RPMI 1640 medium and separately treated with free p53, PGEA/p53, PGAP/p53, and PGHA/p53 complexes for 48 h. As shown in Figure S8, the cell viabilities of PGEA/p53, PGAP/p53, and PGHA/p53 complexes showed a certain decline against A549 cells and H1299 cells, and the cell viability of free p53 gene has almost obvious decline, demonstrating that the cationic vectors can successfully immobilize genes and promote the efficiency of gene transfection. On the contrary, free p53 gene was difficult to penetrate cell membrane because of its large size. All of these results indicate that PGEA, PGAP, and PGHA polycations could efficient promote gene utilization as gene vectors. Moreover, the hydroxyl-rich cationic polymers could shield the positive surface charge to reduce protein aggregation, improve the solubility, and promote the escape of endosome.18, 46 Overall PGHA polycation possesses high gene transfection and the ability of immobilization gene. In the following work, we chose HA to fabricate the polycation for codelivery of anticancer drug and gene.

Synthesis of PCL-ss-P(PEGMA-co-GMA) and PCL-ssP(PEGMA-co-GHA) Copolymers

Figure 8. 1H NMR spectrum of PCL40-ss-P(PEGMA7-co-GMA80) copolymer in CDCl3. The GPC trace of PCL-ss-CPDB and PCL-ss-P(PEGMA-coGMA) in Figure S12. They exhibit unimodal peak distributions with polydispersity indexes (Ð) of 1.20 and 1.60, respectively. And the M̅n values of PCL40-ss-CPDB and PCL40-ss-P(PEGMA7-coGMA80) are 6.0103 g/mol and 1.7104 g/mol. Then, the chemical structure of PCL40-ss-P(PEGMA7-co-GHA80) was characterized by 1H NMR as shown in Figure 9. The peaks (d and e) associated with the epoxy groups of PCL40-ss-P(PEGMA7co-GMA80) disappeared completely. There appears the new chemical shift at δ 2.81 ppm (peak g), indicating that all the oxirane rings were reacted completely. The new peak region located at chemical shifts of δ 3.72 ppm was mainly associated with the methylidyne and methylene protons adjacent to the hydroxyl groups (peak f, −(OH)−CH−CH2−NH− and peak f’, −NH−CH2−CH2−OH). The signal at δ 2.81 ppm was mainly

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than that of the PGHAP nanoparticle, indicating that DOX had been encapsulated into the hydrophobic inner cores.

Figure 9. 1H NMR spectrum of PCL40-ss-P(PEGMA7-co-GHA80) copolymer in DMSO-d6. In addition, PGHAP polycation was also characterized by XPS analysis. As shown in Figure S13, the C1s core-level spectrum could be curve-fitted into three peak components with binding energies (BE’s) at about 284.7 eV, 286.2 eV, and 288.8 eV, attributable to the C−H, C−O, and O=C−O species, respectively. The element contents (C%, N%, and O%) of PCL40ss-P(PEGMA7-co-GHA80) are 70.72%, 4.11%, and 25.17%, respectively. As indicating by the above data, PGHAP have been synthesized successfully.

Self-assembly of PGHAP Copolymer and DOX Encapsulation of PGHAP Nanoparticle Critical aggregate concentration (CAC) is an important design consideration for drug delivery systems, as it reflects the propensity of the molecular building units to aggregate or dissociate in solution state.47 When the concentration of the polymer is higher than the CAC value, the PGHAP polycation can self-assemble into nanoparticles with hydrophobic PCL as the core and hydrophilic P(PEGMA-co-GHA) as the shell in aqueous solution. The CAC value of PGHAP nanoparticles was determined by the steady-state fluorescence probe method using pyrene as the probe. The CAC value of PGHAP nanoparticle is 0.0152 mg/mL as shown in Figure S14.

Figure 10. TEM images and the particle size distribution curves of PGHAP nanoparticles (A and B), and DPGHAP nanoparticles (C and D). The concentration of the nanoparticles was 0.5 mg/mL. The DPGHAP/DNA complexes at various weight ratios were formed by adding same amount of DNA at different concentrations and vortexing for 10 s. The solutions were then allowed to stand at room temperature for 30 minutes. The N/P ratio could be calculated by eqn S7 in the Supporting Information. Zeta potential of the DPGHAP/DNA complexes at different N/P ratios were characterized by DLS measurement. As shown in Figure 11, the zeta potential of DPGHAP/DNA complex is around +30 mV at N/P ratio of 3. It is well known that positive charges of complexes could make polycations/DNA complexes easily be taken up by cells. In addition, a good gene delivery can immobilize effectively and protect DNA in the blood circulation when it reaches tumor cells.

DOX can be encapsulated into the core of PGHAP nanoparticle efficiently, which was named DPGHAP. (The detailed method provided in the Supporting Information section). Moreover, the drug loading content (DLC) and drug loading efﬁciency (DLE) of DPGHAP nanoparticle are calculated by eqs (S4) and (S5), which are 3.67% and 15.2%, respectively.

Physicochemical Property Analysis The morphologies of PGHAP and DPGHAP nanoparticles were observed by TEM analysis. Meanwhile the average particle diameters ( D Z ) and size polydispersity index (size PDI) of nanoparticles were determined by dynamic light scattering (DLS) measurement. For PGHAP and DPGHAP nanoparticles, average diameters of 160 nm and 186 nm were displayed in Figure 10, respectively. The average size of DPGHAP nanoparticle was greater

Figure 11. Zeta potential of DPGHAP/DNA complexes at various N/P ratios. (N/P ratios = 0, 1, 2, 3, 4, 5, and 6)


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attributed to the methylene protons adjacent to the amine groups (g, −CH2−NH−CH2−CH2−NH−CH2−), which confirms that PCL40-ss-P(PEGMA7-co-GHA80) (abbreviated as PGHAP) has been successfully synthesized.



DOI: 10.1039/D0TB01811B


The binding ability of DPGHAP/DNA complex was evaluated by agarose gel electrophoresis at different N/P ratios. The mobility of free DNA could not be observed at N/P ratio of ≥3) in Figure S15(A), that is, DPGHAP/DNA complex had immobilized DNA completely. This result demonstrated that the protonation of the secondary amine groups in DPGHAP could form complexes with negatively charged DNA. Furthermore, in order to verify the stability of the DPGHAP/DNA complexes, we used heparin sodium to simulate the in vivo microenvironment. As shown in Figure S15(B), the mobility of free DNA was completely immobilized in the sample hole when the heparin concentration increased from 0.1 to 1.0 mg/mL, indicating the DPGHAP/DNA complexes could possess good stability in the presence of negative environment.

In vitro DOX Release from DPGHAP Nanoparticle

Figure 13. Cell viabilities of L929 cells, A549 cells, and H1299 cells treated with PGHAP nanoparticle at different concentration for 48 h of incubation.

The release profile of DPGHAP nanoparticle was studied at physiological pH 7.4 and pH 7.4+10 mM GSH media. As shown in Figure 12, the approximately 60% of DOX was released from DPGHAP nanoparticle after incubation for 110 h under pH 7.4+10 mM GSH medium, whereas the release of DOX was about 25% under the condition of pH 7.4. The results confirmed that the DPGHAP nanoparticle possess redox responsive property due to the presence of disulfide bond, which could reduce premature drug release in the bloodstream and promote drug burst release under endo/lysosomal reductive condition in the tumor cells.

Figure 12. In vitro DOX release curves for DPGHAP nanoparticle at pH 7.4 with/without 10 mM GSH. The nanoparticle concentrations were 0.5 mg/mL.

Cytotoxicity and Anti-tumor Assay Cytotoxicity is another important factor to be considered in selecting polymer as drug and gene delivery. The major factor affecting the cytotoxicity of gene vector is charge density of polycations.48 As shown in Figure 13, the cell viability assays demonstrated PGHAP nanoparticle has good biocompatibility against L929 cells, A549 cells, and H1299 cells when the concentration was up to 125 mg/L, which could indicate that PGHAP polycation may be a potential prospect as a drug and gene carriers.

Figure 14. Cell viability of (A) A549 and (B) H1299 cells treated with free DOX, DPGHAP, and DPGHAP/p53 nanoparticles with different concentration for 48 h of incubation. We used the combination therapy of p53 gene and anticancer drug DOX to achieve better therapeutic effects in vitro. MTT assay was used to evaluate the ability of free DOX, DPGHAP, and DPGHAP/p53 nanoparticles to inhibit the proliferation of A549 and H1299 cells. As shown in Figure

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DOI: 10.1039/D0TB01811B

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14(A and B), the half-maximal inhibitory concentration (IC50) values of free DOX, DPGHAP, and DPGHAP/p53 nanoparticles against A549 and H1299 cells were determined to be 1.262 mg L−1, 1.053 mg L−1, 0.364 mg L−1, and 1.037 mg L−1, 0.814 mg L−1, 0.576 mg L−1, respectively. DPGHAP/p53 nanoparticles had the lowest IC50 values for the two different cell lines and used for the other tests. The DPGHAP/p53 nanoparticles can significantly inhibit the growth of tumor cells.


Intracellular Release of DOX and p53 Genes To demonstrate whether the nanoparticles could be efficiently internalized into tumor cells, which could achieve the intracellular drug accumulation and improve gene transfection. The cellular uptake and intracellular drug and gene release behaviors of the DPGHAP/p53-GFP nanoparticles were monitored using a live cell imaging system, which could simultaneously record the dynamic uptake process and the variation in the fluorescence intensity inside the A549 cells.

Figure 15. Live cell imaging system images of A549 cells incubated with (A) DPGHAP/p53-GFP nanoparticles and (B) same quantity of free DOX and p53 gene (DOX dosage was 7.5 mg/L, p53-GFP gene dosage was 6 mg/L, N/P ratio was 6) for different times. For each panel, images from left to right show the cell nuclei stained with H 33342 (blue), the GFP fluorescence in cell (green), the DOX fluorescence in cells (red), and overlays of the red/blue/green images. The scale bars are 50 μm in all images. As shown in Figure 15(A), there are neither red nor green fluorescence around the nucleus at the beginning of incubation with A549 cells. Then, the red and green fluorescence in cytoplasm could be observed when A549 cells were treated with DPGHAP/p53-GFP nanoparticles for 6 h. We can observe that the intensity of fluorescence increased gradually and the stronger fluorescence appeared in the nucleus. After 24 h of incubation, the results demonstrated that most of the DPGHAP/p53-GFP nanoparticles were internalized by A549 cells via endocytosis. In contrast, the fluorescence intensity of A549 cells that were incubated with along free DOX and p53-GFP was weak at 24 h, as shown in Figure 15(B), from

which we cannot almost observe the red and green fluorescence around the nucleus because the DOX enters into tumorous cells by a diffusion process and passes through the cell membrane through a concentration gradient.49, 50, 51 In addition, free DOX is easy to escape from the tumorous cells by efflux pump. Free p53-GFP gene would be generally decomposed directly, and unable to replicate, survive stably and express itself when enters cells. Hence, there are stronger red and green fluorescence intensity incubated with DPGHAP/p53-GFP nanoparticles than that of free DOX and p53-GFP. All of these results confirmed that DPGHAP/p53-GFP nanoparticles could promote the transmembrane transport and endosome escape, improve the drug release and gene transfection in the tumor cells.

Conclusions In the first part of this work, we prepared four kinks of polycations (PGEA, PGAP, PGDA and PGHA) containing hydroxyl and amino groups via RAFT copolymerization and postmodification method, and demonstrated that these polycations possess good biocompatibility and high gene transfection. Among them, the PGHA polycation has higher gene transfection efficiency than the other species. In the second part, based on PGHA, we synthesized an amphiphilic copolymer PCL-ss-P(PEGMA-co-GHA) by a combination of ROP and RAFT copolymerization. The polycation could self-assemble into nanoparticles (abbreviated as PGHAP), and encapsulate anticancer drug DOX to form DOX-loaded nanoparticles (namely DPGHAP). The size of PGHAP and DPGHAP nanoparticles were 160 nm and 186 nm, respectively. DOX could be released from DPGHAP nanoparticles, the cumulative release amount was up to 60% under the reductive medium. In addition, the in vitro MTT assay results confirmed that the PGHAP nanoparticles possess good biocompatibility. The DPGHAP/p53-GFP complexes could superior antitumor effects against A549 and H1299 cells with IC50 of 0.364 mg/L and 0.576 mg/L. Moreover, the DPGHAP/p53-GFP nanoparticles could co-deliver p53 gene and anticancer drug DOX into A549 cells by endocytosis. Therefore, the constructed cationic polymer as a co-vectors of anticancer drugs and genes will have a promising application prospect.

Conflicts of interest The authors declare no competing financial interest.

Acknowledgements We gratefully think the financial supports from the National Natural Science Foundation of China (21975169 and 21374066), the Major Program of the Natural Science Project of Jiangsu Higher Education Institutions (15KJA150007), a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and SoochowWaterloo University Joint Project for Nanotechnology from Suzhou Industrial Park. We are also grateful to Professor Jian Liu (FUNSOM, Soochow University) for his valuable help in the cell-related tests.

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DOI: 10.1039/D0TB01811B


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