Synthesis of Diblock/Statistical Cationic Glycopolymers with Pendant Galactose and Lysine Moieties: Gene Delivery Application and Intracellular Behaviors

New series of cationic block copolymers PHML-b-PMAGal and statistical copolymers P (HML-st-MAGal) with pendant natural galactose and (l-)-lysine moieties were prepared via RAFT (reversible addition-fragmentation chain-transfer) polymerization. The block/statistical copolymers showed high plasmid DNA binding affinity (N/P<2) and the as-formed polyplexes were spherical nanoparticles with the average size of 100~300 nm and surface zeta potentials of +30.2~+46.3 mV. The cytotoxicity and gene transfection efficacy of the PHML-b-PMAGal and P (HML-st-MAGal) vectors strongly depended on the polymer architectures (block/statistical) and galactose contents. Notably, the statistical copolymer P(HML40-st-MAGal4) with 4.8% galactose content showed the highest gene transfection efficiency among the synthesized cationic polymers, 6.8-folds higher than that of the “gold standard” bPEI-25K in the presence of 10% FBS (Fetal bovine serum) in various cell lines. Intracellular uptake mechanism (with 10% FBS) study demonstrated that the P(HML40-st-MAGal4)/pDNA polyplexes mainly entered into H1299 cells through caveolaemediated endocytosis and microtubule-dependent endocytosis pathway. Moreover, the fluorescent imaging study showed the P(HML40-st-MAGal4)/pDNA polyplexes possessed obvious “lysosomal escaping” effect that led to efficient pDNA release, which might interpret the fact of the significant increase of the related gene transfection efficiency. Moreover, it could be anticipated that the P(HML40-st-MAGal4) cationic glycopolymer might be employed as low toxic, high efficient and serum-compatible gene carriers for practical application.


Introduction
In the past few decades, cationic polymeric non-viral gene vectors have been developed as a new technique for gene therapy, and it is well known that successful gene therapy largely depends on the choice of suitable building blocks in construction of the cationic polymeric gene vectors [1][2][3][4] .Although many cationic polymeric vectors were developed to mediate efficient intracellular gene transfection, some defects such as high cytotoxicity 5 , immunogenicity 6 and low serum-compatibility 7, 8 severely restricted their clinical application in gene therapy.To solve these issues, a general approach in previous works is to conjugate/modify cationic polymers with polyethylene glycol (PEG), which could be employed to shield cationic surface charge, decrease cytotoxicity and improve serumcompatibility of cationic polymer vector/gene complexes 5,9 .However, it should be noted that the "stealth" effect of PEG could decrease cell adhesion and intracellular uptake, further resulting in lower transfection efficiency of the polyplexes 10 .Moreover, the PEG-modified polymeric vectors might induce immune-response as antigens and would be erased by macrophage systems after injection 11 .
Recently, several biocompatible building blocks such as zwitterionic molecules 12,13 and carbohydrates 14 have been developed as alternative substitutes to PEG moieties in gene/drug delivery systems.Among them, carbohydrates are highly biocompatible natural products with good hydrophilicity and biodegradable manners.It had been disclosed that cationic glycopolymer vectors constructed by carbohydrates and cationic units exhibited low cytotoxicity and serum-resist stability, which resembled that of the PEG-conjugated polycations due to the positive charge shielding effect.But unlike PEG, the carbohydrates units in glycopolymers have the advantage of enhancing pDNA binding affinity through hydrogen bond interactions, which may further facilitate high gene transfection efficiency [15][16][17][18] .Besides, the multi-hydroxyl groups on carbohydrate units offered them the possibility for further functional derivation/modification 19,20 .e. g.Azzam et al 21 synthesized some polyethylenimine and spermine modified dextran and branched-starch cationic polymers, found the gene transfection capability largely relied on the polysaccharide structures, and dextran-derived cationic polymers showed the highest transfection capability.Davis et al 22 reported that the length of cationic blocks played essential roles in the cytotoxicity and gene transfection of cyclodextrin-based cationic gene vectors.Gu et al 23 prepared (l)-lysine-modified chitosan polymers, complicated and uncontrollable structures of these natural polysaccharide glycopolymers, it is difficult to elucidate their concise structure-activity relationships.Thus, developing new cationic glycopolymers with defined and controllable molecular structures as efficient gene vectors/carriers is of high demand.
With the development of controlled free radical polymerization, a lot of functional glycopolymers with well-defined structures and controllable molecular-weight could be facilely prepared 24,25 , which made it possible for further investigating the correlation between polymer structure and gene transfection related properties.e. g.Narain et al synthesized a series of well-defined block and random glycopolymers as gene vectors and found the latter showed higher gene transfection efficiency than the former one [26][27][28][29] .Reineke et al prepared some cationic glycopolymers as gene vectors and disclosed that introducing of sugar-containing blocks could increase the stability of pDNA-loaded complexes by preventing their aggregation in culture medium 14,[30][31][32][33] .However, to our knowledge, many cationic building blocks used for constructing glycopolymeric gene vectors were non-biodegradable or low-biocompatible.Alternatively, cationic amino acids (such as (l)-lysine and (l)-arginine) serve as essential biocompatible natural cationic building blocks for constructing new cationic gene vectors with low cytotoxicity and high pDNA binding affinity 34,35 .Moreover, some cationic glycopolymers constructed by amino acids and carbohydrates could act as high efficient synthetic mimics of natural virus-based gene vectors 36,37 .However, up to now, design and synthesis of well-defined cationic amino acid-based glycopolymer gene carriers with low cytotoxicity, high gene transfection efficiency and preferred serum-compatibility is still a challenge.Besides, the structure-activity relationships and intracellular features (especially precise mechanisms of intracellular uptake and trafficking) of the amino acid-based glycopolymer gene carrier systems are still far from clear and need to be deeply investigated.
To develop new amino acid-based cationic glycopolymers with well-defined structures and elucidate the structure-dependent gene transfection properties, in this work, a series of diblock (PHML-b-PMAGal) and statistical (P(HML-st-MAGal)) cationic glycopolymers with pendant galactose and (l)-lysine moieties were synthesized by RAFT polymerization.The cytotoxicity, in vitro luciferase (Luc) gene transfection efficacy and the impact of serum on the gene transfection efficiency were evaluated in various cell lines.Finally, the cellular uptake/endocytosis pathway and
The as-prepared PHMLBoc, PHMLBoc-b-PMAIGal and P(HMLBoc-st-MAIGal) polymer precursors were deprotected at room temperature in mixed trifluoroacetic acid (TFA) and tetrahydrofuran (THF) (2/1, v/v) for 24 h, and then precipitated in cold diethyl ether, filtrated and dried in vacuum, the final cationic polymer products were obtained as white solids.
PHML: NMR spectrum: 1 H NMR spectrum were conducted on a Varian-300 FT-NMR spectrometer at 300.0 MHz for proton nuclei, and 13 C NMR was implemented on a Bruker Avance-400 NMR spectrometer, operating at 100.0 MHz for 13 C nuclei, tetramethylsilane (TMS) was used as internal chemical shift reference in all NMR measurements.
FT-IR spectra: FT-IR spectra were recorded on a Bio-Rad FTS-185 spectrometer at room temperature in the wavelength ranging from 4000 to 500 cm -1 with 4 cm -1 spectral resolution (64 times scanning).

Gel permeation chromatography (GPC):
Average molecular weights (M n and M w ) and polydispersity index (M w /M n ) of the glycopolymer precursors were measured at 35°C on a PerkinElmer 200 GPC equipped with a refractive index detector (RI).Tetrahydrofuran was utilized as the eluent at a flowing rate of 1.0 mL/min, and a series of commercial polystyrene standards (Polymer laboratories, 2.4.Agarose gel electrophoresis pDNA binding affinity of the PHML, PHML-b-PMAGal and P(HML-st-MAGal) cationic polymers were determined by agarose gel electrophoresis.Each cationic polymer/pDNA polyplexes were prepared by mixing cationic polymers with 1 µg pDNA at various N/P charge ratios in deionized water.After 30 min incubation at 37°C, the mixed solution was loaded onto agarose gel (1.2% w/v) containing ethidium bromide (0.1 mg/ml in gel) in TAE buffer solution, commercially available DNA markers and naked pDNA were used as controls.The electrophoresis was run at 100 V for 40 min.Finally, the pDNA retardation was observed and recorded using a UV transilluminator (UVP, USA).

Transmission electronic microscopy (TEM)
The morphology of the PHML 40 /pDNA, PHML 40 -b-MAGal 3 /pDNA and P(HML 40 -st-MAGal 4 )/ pDNA polyplexes were measured by TEM (JEOL-1230, JEOL Co. Ltd, Japan) with an acceleration voltage of 80 KV.For each sample preparation, the polyplex solution (1.0 mg/mL) was dropped onto a 300-mesh carbon-coated copper grid, excess solution was absorbed and removed with filter paper, and the grid was then air-dried at room temperature.

MTT cytotoxicity assay
In vitro cytotoxicity of the PHML, PHML-b-PMAGal and P(HML-st-MAGal) cationic polymers was evaluated by MTT assay in H1299 cells.First, H1299 cells were seeded into 96-well microplates at a density of 5.0×10 3 cells/well in 100 µL of RPMI-1640 medium containing 10% FBS.After 24 h incubation, the culture medium was replaced with fresh RPMI-1640 medium (100 µL, with 10% 24 h.Then 20 µL of MTT solution (5 mg/mL) was added into each well and incubated for additional 2 h at 37°C.After that, the medium was discarded and 100 µL/well DMSO was added with mildly shaken for 10 min to dissolve the formed MTT formazan.The absorbance of all samples was measured with six replicates (n=6) by using microplate reader (BioTek, ELx800, USA) at λ=490 nm (λ=630 nm as the reference wavelength).Relative cell viability (%) was calculated according to the equation: Relative cell viability (%) = (OD490 sample -OD630 sample )/(OD490 control -OD630 control ) × 100%

In vitro luciferase gene transfection assay
For in vitro luciferase gene transfection assay of the PHML, PHML-b-PMAGal and P(HML-st-MAGal) cationic polymers, first, H1299 cells were seeded in 24-well plates (4×10 5 cells/well) with 500 µL of RPMI-1640 medium containing 10% FBS.After incubation for 24 h, the medium were replaced with 500 µL fresh RPMI-1640 medium (10% FBS) containing the polyplex (1 µg pDNA/well) at various charge ratios and further incubated for 24 h at 37 °C, the commercially available bPEI-25k (N/P=10) was used as the control.The luciferase assay was performed according to the protocols and relative light units (RLUs) were measured with the Promega GloMax®-Multi Single Tube Multimode Reader (E6070, USA), protein quantification was determined by BCA assay kit (Applygen Technologies Inc, China) according to the protocol, and relative light unit per milligram of Luciferase protein (RLU/mg) was calculated to evaluate the cell transfection efficacy of the cationic polymers (n=3).
Then the H1299 cells were washed with 1×PBS for three times to eliminate fluorescence background.
The cells were fixed with 4% paraformaldehyde in 0.12 M phosphate buffer (pH=7.4) for 10 min and washed with 1×PBS for three times.Lysotracker (final working concentration 100 ng/mL) was added to stain the endosome/lysosome and incubated for 15 min before fluorescence imaging.
Finally, the intracellular localization of the P(HML 40 -st-MAGal 4 )/Cy3-pDNA polyplexes were visualized and recorded on a Nikon Ti-S invert fluorescence microscopy.

Preparation and characterization of the cationic polymers
In order to prepare new cationic glycopolymers with the merit of well-defined, functionalized and controllable structures, and to further study the effect of the functional building blocks and glycopolymers structures (block vs statistical) on the related physico-chemical and biological properties, a homopolymer PHML with pendent (l)-lysine, and series of block glycopolymers PHML-b-PMAGal and statistical glycopolymers P(HML-st-MAGal) with pendant galactose and (l)-lysine groups/moieties were prepared via RAFT polymerization (Scheme 1).First, HEMA-Boc-Lys monomer was synthesized according to our previous work 39 , the glycomonomer 6-O-methacryloyl-1,2,3,4-di-O-isopropylidene-galactopyranose (MAIGal) was prepared according to the literature 40  HEMA-Boc-Lys monomers with predetermined feeding ratios.The structures of these polymers were characterized by 1 H NMR (Figure 1) and GPC (Figure 2).As shown in Figure 1, the average degree of polymerization of the MAlGal monomers DP (MAIGal) was calculated via the formula: DP decomposed after TFA-deprotection [41] , so the dithiobenzoate end groups were removed and replaced with an asterisk *).Taking P(HML 40 -st-MAGal 13 ) as an example, the 1 H NMR and FTIR characterization were shown in Figure S2 and Figure S3, respectively.It could be seen that the strong peaks at δ=1.36~1.42ppm (acetal and Boc protecting groups) remarkably decreased (Figure S2), and the amide proton signal at 6.6 and 7.0 ppm disappeared along with the appearance of the new protonated amine signal at 8.1 and 8.8 ppm, which indicated the complete removal of the acetal and Boc protecting groups.In Figure S3, the stretch vibration signal of methyl group for the acetal and Boc protecting groups (ν C-CH3 ) at 1366 cm -1 disappeared along with the appearance of the hydroxyl group signal (ν O-H ) at 3431 cm -1 , further confirming the structure of the P(HML 40 -st-MAGal 13 ).The results demonstrated the successful preparation of the cationic glycopolymers.

pDNA binding affinity of the glycopolymers by agarose gel electrophoresis
pDNA binding affinities of the synthesized cationic diblock glycopolymers (PHML-b-PMAGal) and statistical glycopolymers (P(HML-st-MAGal)) were evaluated by agarose gel electrophoresis with the cationic homopolymer PHML as the control.As shown in Figure 3, all the cationic polymers were able to retard pDNA completely at the N/P charge ratio of 1, which might due to the strong pDNA binding affinity of the primary amino groups (pK a =10.8) on the (l)-lysine moiety 42 .
Noteworthy, different from some PEG-containing cationic polymers, introduction of galactose moieties into poly-(l)-lysine cationic polymers did not reduce the pDNA binding affinity, which might be partially attributed to the hydrogen bond-mediated interactions between the galactose moiety and pDNA 43 .Moreover, the results indicated that the PHML-b-PMAGal and P(HML-st-MAGal) cationic glycopolymers possessed high pDNA binding affinity, which made them as efficient cargoes for pDNA loading.

Average particle size and zeta potential measurements
Average particle size and zeta potential of polymers/pDNA polyplexes were essential factors for cytotoxicity, cellular uptake and gene transfection efficiency 34 .Herein, the average particle sizes of the cationic glycopolymers/pDNA polyplexes at various N/P charge ratios were measured by DLS with the homopolymer PHML as a control.As shown in Figure 4, all the polyplexes displayed

Journal of Materials Chemistry B Accepted Manuscript
PHML 40 -b-PMAGal 15 /pDNA and P(HML 40 -st-MAGal 13 )/pDNA (N/P=40) polyplexes, which containing higher percentage of galactose moieties (16.2% and 14.3%, respectively), showed comparatively larger particle size than the other polyplexes, that might due to the formation of a galactose-containing hydrophilic shell/corona on the surface of polyplex payloads by the strong hydration effect of galactose moieties.Moreover, morphology of the PHML 40 /pDNA, PHML 40 -b-MAGal 3 /pDNA and P (HML 40 -st-MAGal 4 )/pDNA polyplexes was examined by transmission electron microscopy (TEM).As shown in Figure S4, all of the polyplexes formed spherical-shaped nanoparticles with the size range of 30~110 nm, which was smaller than the size measured by DLS, due to the shrinkage of the nanoparticles under drying process during the preparation of TEM samples 35,44 .Besides, the zeta potentials of the polyplexes were measured by DLS.As shown in Figure 4b, the polyplexes displayed positive surface potentials (+30.2~+46.3mV) at N/P charge ratios from 10 to 100, for each polyplexes, the zeta potential almost maintained at the same level and showed less dependence of the N/P charge ratios (N/P 10~100), suggesting the complete pDNA binding of the cationic polymers within the N/P range, due to the strong electrostatic interactions between the cationic lysine groups/moieties with pDNA, which is in accordance with the above electrophoresis results and our previous work 39 .Moreover, the relatively small particle size and positive surface potential of the polyplexes may benefit their cell surface adhesion and following intracellular uptake process 36 , which may further improve the intracellular gene transfection capability.

Cytotoxicity
Cytotoxicity is one of the most important parameters for evaluating the safety of the non-viral gene vectors toward therapeutic application.Recently, it has been revealed that introducing of some natural moieties such as cholesterol, sugar and amino acids could improve biocompatibility and decrease cytotoxicity of polymeric gene vectors 34 .Herein, the cytotoxicity of cationic polymers PHML, PHML-b-PMAGal and P(HML-st-MAGal) were measured by MTT assay with the commercially available bPEI-25K as a control.As shown in Figure 5, severe cytotoxicity (relative cell viability, RCV<30%) was observed in H1299 cells when incubated with bPEI-25k at the dose up to 60 µg/mL, whereas PHML, PHML 40 -b-PMAGal 3 and P(HML 40 -st-MAGal 4 ) showed lower cationic polymers PHML 40 -b-PMAGal 15 and P(HML 40 -st-MAGal 13 ) exhibited much lower cytotoxicity (RCV>80%) even at high concentration of 200 µg/mL, which indicated that introducing of biocompatible galactose moieties could decrease the cytotoxicity of cationic polymers.Similarly, previous literatures had reported sugar (cyclodextrin)-conjugation on some cationic dendrimer gene carriers could lower their cytotoxicity 45 .Moreover, the MTT results suggested that the cationic glycopolymers might be employed as comparatively safe vectors for gene delivery application.

In vitro luciferase gene transfection
In vitro gene transfection activity of PHML, PHML-b-PMAGals and P(HML-st-MAGal)s cationic polymers were evaluated under the presence of serum (10% FBS) in H1299 cells, with commercially available "gold standard" bPEI-25K as the control.As shown in Figure 6, homopolymer PHML showed its optimum transfection efficiency of 4.5-fold higher than that of bPEI-25K, the low-galactose-content cationic glycopolymers PHML 40 -b-PMAGal 3 and P(HML 40 -st-MAGal 4 ) showed their highest transfection efficiency at the N/P ratio of 40, 1.4~6.8-foldhigher than the transfection level of the bPEI-25k (**P<0.05).However, the high-galactose-content cationic glypolymers PHML 40 -b-PMAGal 15 and P(HML 40 -st-MAGal 13 showed comparatively lower transfection efficiencies than their low-galactose-content counterparts.The lower gene transfection efficiency of the high-galactose-content cationic polymers might due to the surface charge shielding effect of their excess galactose moieties, which hindered the effective adhesion of the cationic lysine moieties with negatively-charged cell membrane, thus further decreased the cellular uptake capability and subsequent gene expression.Noteworthy, the statistical glycopolymer P(HML 40 -st-MAGal 4 ) showed higher transfection capability than the block glycopolymer PHML 40 -b-PMAGal 3 , which have similar chemical component but different copolymer sequence/ arrangement, indicating that the gene transfection efficiency depends to some extent on the copolymer sequence/arrangement of the cationic glycopolymers.Similarly, Ahmed et al 29 observed similar transfection trend on a series of glucose-containing cationic glycopolymers, they supposed that the gene transfection difference might be attributed to the different interactions between the sequence-varied cationic glycopolymers and negatively-charged serum proteins.
To further evaluate the practical applications of the cationic glycopolymers in different cell lines under serum conditions (10% FBS), based on the transfection results in H1299 cells, the optimized

Journal of Materials Chemistry B Accepted Manuscript
statistical cationic glycopolymer P(HML 40 -st-MAGal 4 ) polyplexes (N/P=40) was utilized as the transfection model.As shown in Figure 7, the P(HML 40 -st-MAGal 4 ) polyplexes showed obviously higher luciferase gene transfection efficiency (4.8~87-fold) than the "gold standard" bPEI-25K in SK-HEP-1, MCF-7, CHO, HeLa and COS-7 cells.The results suggested that the statistical cationic glycopolymer P(HML 40 -st-MAGal 4 ) might be employed as a serum-compatible and high efficient transfection agent for cellular gene delivery and practical gene therapy.
3.6.Endocytosis mechanism/pathway of the P(HML 40 -st-MAGal 4 )/pDNA polyplexes It had been revealed that gene/drug delivery efficiency of nano-delivery systems greatly relied on their endocytosis mechanism/pathway 46 , to our knowledge, the study on endocytosis pathway of cationic glycopolymer vectors/pDNA complexes are still rare.Herein, in order to elucidate the endocytosis pathway of the P(HML 40 -st-MAGal 4 )/pDNA polyplexes (N/P=40, optimized condition) under serum environment (10% FBS), we investigated the luciferase expression in the presence of various endocytosis-specific inhibitors 47 .The H1299 cells transfected with P(HML 40 -st-MAGal 4 )/ pDNA polplexes (N/P=40) in the absence of the inhibitors was set as the control (luciferase expression: 100%).As shown in Figure 8, the luciferase expression of the P(HML 40 -st-MAGal 4 )/ pDNA polyplexes obviously decreased with the addition of M-β-CD (62.3 %) and nocodazole (18.2 %) respectively, indicating the P(HML 40 -st-MAGal 4 )/pDNA polplexes entered into H1299 cells mainly through the Caveolae-mediated (or lipid-raft-mediated) endocytosis and microtubuledependent pathway.Interestingly, with addition of the clathrin-mediated endocytosis inhibitor chlorpromazine, the luciferase tranfection increased to around 150% (1.5-fold).Breackmans et al 48 also reported similar results, which might be attributed to the proton-buffering effect of chlorpromazine.The related mechanism in detail is still not very clear until now.Moreover, the luciferase expression of the H1299 cells incubated with the P(HML 40 -st-MAGal 4 )/pDNA polplexes decreased to 86.2% with addition of macropinocytosis inhibitor amiloride, which indicated that the macropinocytosis might not be served as the dominant endocytosis pathway.In previous works, we disclosed that a series of arginine-bearing polymers with high gene transfection efficiency undergoes caveolae (lipid-raft) mediated endocytosis pathway and followed by endosome localization 47 .
Similarly, Helenius et al 49 revealed that caveosome-endoplasmic reticulum localization was followed by caveolae-mediated endocytosis of the cationic cargoes.Thus, we deduced that the caveolae-mediated (or lipid-raft-mediated) endocytosis pathway may contribute to the high transfection efficiency of the P(HML 40 -st-MAGal 4 )/pDNA polyplexes.3.7.Intracellular trafficking and localization of the P(HML 40 -st-MAGal 4 )/pDNA polyplexes In order to further explore the transfection mechanism, the intracellular trafficking and localization of the P(HML 40 -st-MAGal 4 )/Cy3-pDNA polyplexes under the presence of serum (10% FBS) were investigated by fluorescence microscopy in H1299 cells, and the endosome/lysosome of H1299 cells was stained by lysotracker kit 50 .As shown in Figure 9 (merged image), after transfection for 2 h, it could be observed that the red fluorescence of P(HML 40 -st-MAGal 4 )/Cy3-pDNA polyplexes were overlapped with the green fluorescence of lysotracker and resulted in obvious yellow fluorescence emission, indicating the polyplexes were entrapped in endosome/lysosome.After 6h incubation, the yellow fluorescence almost disappeared and the red and green fluorescence dots clearly separated, which indicated successful and efficient "lysosome escape" effect.The results might be attributed to the proton buffering effects of cationic (l)-lysine moieties, which was regarded as one of the crucial factors to achieve efficient gene transfection.Moreover, some of the polyplexes could be found distributed inside the H1299 cell nuclei (Figure S5).Recently, it has been reported that internalized sugar-containing nanoparticles demonstrated "Trojan horse effect" and be able to colocalized within various subcellular organelles such as endosome/lysosome, endoplasmic reticulum, Golgi apparatus, and so on 51 .Based on this, we expected that the cationic glycopolymers with certain sugar-moieties may possess sugar-mediated intracellular trafficking/ localization effect, which may further provide new approaches for improving gene/drug delivery efficacy by directing gene/drug payloads go through the probably efficient "sugar-mediated pathways".In addition, further research on the correlation between sugar/carbohydrate moieties and intracellular localization/trafficking manners of the glycopolymer-based gene/drug delivery systems is currently under investigation in our lab.

Conclusions
In summary, a series of block/statistical cationic glycopolymers PHML-b-PMAGal and P(HML-st-MAGal) with pendant (l)-lysine and galactose moieties were prepared via RAFT polymerization and following Boc-deprotection.All the synthesized cationic glycopolymers showed diameter (150~300 nm) and positive zeta potential (+30~45 mV) of the cationic glycopolymers/pDNA polyplexes were found suitable for intracellular uptake.MTT assay showed that the cationic glycopolymers had significantly lower cytotoxicity than bPEI-25K, which depended on the block/statistical polymer architecture and galactose contents.Moreover, it could be noticed that the statistical copolymer P(HML 40 -st-MAGal 4 ) with 4.8% galactose content showed the highest gene transfection efficiency among the synthesized cationic glycopolymers in the presence of 10% FBS, much higher than the "gold standard" bPEI-25K in various (H1299, SK-HEP-1, MCF-7, CHO, HeLa and COS-7) cell lines.Endocytosis pathway analysis indicated that the P(HML 40 -st-MAGal 4 )/pDNA polyplexes entered into H1299 cells mainly through the caveolae-mediated (or lipid-raft-mediated) endocytosis pathway and greatly depended on microtubule.Moreover, the P(HML 40 -st-MAGal 4 )/pDNA polyplexes showed related fast cellular uptake capability and obvious endosome/lysosome escaping effect.In conclusion, the current results suggested the synthesized cationic glycopolymers might be served as potential candidates for safe and efficient gene delivery in practical applications.

Journal of Materials Chemistry B Accepted Manuscript
. The synthesis routes and characterization of the monomers were shown in supporting information (Scheme S1 and Figure S1).The homopolymer PHMLBoc 40 was obtained as a precursor via RAFT polymerization of HEMA-Boc-Lys monomer by using AIBN/4cyanopentanoic acid dithiobenzoate system.Then MAIGal monomer was further polymerized by employing PHMLBoc 40 precursor as a Macro-chain-transfer-agent (MacroCTA) and two diblock glycopolymers containing different galactose ratios (PHMLBoc 40 -b-PMAIGal 3 and PHMLBoc 40 -b-PMAIGal 15 ) were obtained by adjusting the feeding ratio of MAIGal monomers and PHMLBoc 40 MacroCTA.For comparison, two statistical glycopolymers P(HMLBoc 40 -st-MAIGal 4 ) and P(HMLBoc 40 -st-MAIGal 13 ) were prepared through RAFT polymerization of MAIGal and