PEGylated Amine-Functionalized Poly(ε-caprolactone) for the Delivery of Plasmid DNA

As a promising strategy for the treatment of various diseases, gene therapy has attracted increasing attention over the past decade. Among various gene delivery approaches, non-viral vectors made of synthetic biomaterials have shown significant potential. Due to their synthetic nature, non-viral vectors can have tunable structures and properties by using various building units. In particular, they can offer advantages over viral vectors with respect to biosafety and cytotoxicity. In this study, a well-defined poly(ethylene glycol)-block-poly(α-(propylthio-N,N-diethylethanamine hydrochloride)-ε-caprolactone) diblock polymer (PEG-b-CPCL) with one poly(ethylene glycol) (PEG) block and one tertiary amine-functionalized cationic poly(ε-caprolactone) (CPCL) block, as a novel non-viral vector in the delivery of plasmid DNA (pDNA), was synthesized and studied. Despite having a degradable polymeric structure, the polymer showed remarkable hydrolytic stability over multiple weeks. The optimal ratio of the polymer to pDNA for nanocomplex formation, pDNA release from the nanocomplex with the presence of heparin, and serum stability of the nanocomplex were probed through gel electrophoresis. Nanostructure of the nanocomplexes was characterized by DLS and TEM imaging. Relative to CPCL homopolymers, PEG-b-CPCL led to better solubility over a wide range of pH. Overall, this work demonstrates that PEG-b-CPCL possesses a range of valuable properties as a promising synthetic vector for pDNA delivery.


Introduction
The first clinical study on gene therapy was conducted at the National Institutes of Health (NIH) in 1989, and it demonstrated that genetically modified human cells could be transferred to patients in a feasible and safe manner [1]. Following this study, almost 2600 clinical trials on gene therapy have been initiated or performed worldwide through November 2017 [2]. Thus far, six gene delivery products have been approved by the U.S. Food and Drug Administration (FDA), European Marketing Authorization (EMA), and the China Food and Drug Administration (CFDA), indicating steady development and growing confidence in gene therapy [2]. enables the polymer to form polyplexes with genetic material, mainly in the CPCL-based interior domain of the polyplex nanostructure while protecting nucleic acid cargo from degradation by serum endonucleases and evading immune detection. Moreover, because the tertiary amine groups of CPCL block can adsorb protons within endosomes to induce proton sponge effect, the intracellular release of polyplexes after endocytosis can be facilitated [26]. On the other hand, the hydrophilic PEG block of the PEG-b-CPCL diblock copolymer results in a PEG-based exterior shell domain of the polyplex nanostructure, leading to improved aqueous solubility and colloidal stability as well as increased circulation time of the polyplexes through minimizing nonspecific interactions with serum components. Figure 1 demonstrates how PEG-b-CPCL forms polyplexes with pDNA and facilitates the transfection process upon administration.
Materials 2020, 13, x FOR PEER REVIEW 3 of 17 protecting nucleic acid cargo from degradation by serum endonucleases and evading immune detection. Moreover, because the tertiary amine groups of CPCL block can adsorb protons within endosomes to induce proton sponge effect, the intracellular release of polyplexes after endocytosis can be facilitated [26]. On the other hand, the hydrophilic PEG block of the PEG-b-CPCL diblock copolymer results in a PEG-based exterior shell domain of the polyplex nanostructure, leading to improved aqueous solubility and colloidal stability as well as increased circulation time of the polyplexes through minimizing nonspecific interactions with serum components. Figure 1 demonstrates how PEG-b-CPCL forms polyplexes with pDNA and facilitates the transfection process upon administration.

Measurements
1 H NMR measurements were conducted at 500 MHz using a Varian INOVA-500 (Varian, Inc., Palo Alto, CA, USA). The temperature of all measurements were set at 25 °C and tetramethylsilane (TMS) was used as an internal reference. VnmrJ software (v. 4.2, Agilent Technologies, Inc., Santa Clara, CA, USA) with a university license was used to analyze the raw data.
Hydrodynamic diameter (Dh) and zeta potential (ζ) of polymer:pDNA nanocomplexes were analyzed by using dynamic light scattering (DLS). The instrument was a Zetasizer nano-ZS90 (Malvern Panalytical, Malvern, UK), with a 4 mW 633 nm HeNe laser as the light source. All experiments were conducted at a fixed measuring angle of 90° to the incident laser beam and 25 °C. Zetasizer Nano software (v. 7.11, Malvern Panalytical, Malvern, UK) was used to analyze the raw data for both Dh and ζ.
Transmission electron microscopy (TEM) images were taken using a JEOL 2010 microscope (JEOL Ltd., Akishima, Japan). TEM samples were prepared using 400 mesh carbon-coated copper grids. Sample solutions with DLS count rate of ~250 kcps in water were dip coated onto the TEM grids. Then the water was completely dried under vacuum and no staining was applied for the samples.

Measurements
1 H NMR measurements were conducted at 500 MHz using a Varian INOVA-500 (Varian, Inc., Palo Alto, CA, USA). The temperature of all measurements were set at 25 • C and tetramethylsilane (TMS) was used as an internal reference. VnmrJ software (v. 4.2, Agilent Technologies, Inc., Santa Clara, CA, USA) with a university license was used to analyze the raw data.
Hydrodynamic diameter (D h ) and zeta potential (ζ) of polymer:pDNA nanocomplexes were analyzed by using dynamic light scattering (DLS). The instrument was a Zetasizer nano-ZS90 (Malvern Panalytical, Malvern, UK), with a 4 mW 633 nm HeNe laser as the light source. All experiments were conducted at a fixed measuring angle of 90 • to the incident laser beam and 25 • C. Zetasizer Nano software (v. 7.11, Malvern Panalytical, Malvern, UK) was used to analyze the raw data for both D h and ζ.
Transmission electron microscopy (TEM) images were taken using a JEOL 2010 microscope (JEOL Ltd., Akishima, Japan). TEM samples were prepared using 400 mesh carbon-coated copper grids. Sample solutions with DLS count rate of~250 kcps in water were dip coated onto the TEM grids. Then the water was completely dried under vacuum and no staining was applied for the samples.
Toluene was refluxed over CaH 2 for 2 h prior to distillation. THF was dried by distillation over sodium-benzophenone when the solution was dark blue. Water content of mPEG 45 -OH was removed by two times of azeotropic distillation in dry toluene at 175 • C. HMPA and CL were distilled from CaH 2 shortly before use. LDA, as a pyrophoric material that is highly flammable, water reactive, toxic, corrosive and known to be carcinogenic and teratogenic, was used according to strict safety guidelines. Deionized water, obtained from a Thermo Scientific Barnstead Nanopure ultra-pure water system, was used for the preparation of all buffer solutions. All other chemicals were used without further purification.

Synthesis of Allyl-Functionalized CL (ACL, 1)
ACL 1 was synthesized according to a previously reported procedure (Scheme 1) [30]. Briefly, 150 mL of freshly distilled dried THF was air-free transferred to a vacuum dried 250-mL reaction flask with a magnetic stirring bar under vigorous nitrogen purging and cooled in a dry ice/acetone bath. Upon reaching a solution temperature of −78 • C, the LDA solution (2.0 M, 25 mL) was carefully added dropwise to the reaction flask. After stirring for a few minutes, a diluted solution of CL (5.00 g) in THF (30 mL) was added slowly over a period of 1 h. This mixture was stirred at −78 • C for another 1 h, and allyl bromide (6 mL) in HMPA (10 mL) was added dropwise. The reaction system was allowed to warm up over 1 h to −45 • C and was kept at this temperature for another 1.5 h. The reaction was quenched by adding a saturated NH 4 Cl solution to the flask. The reaction mixture was allowed to warm up to room temperature and then washed with saturated NaCl and NH 4 Cl solutions (3×). The upper organic layer was concentrated and added to cold diethyl ether. The white precipitate was removed by filtration, and the filtrate was dried over magnesium sulfate and concentrated under vacuum. The resulting crude product was purified through a silica gel column chromatography eluted with hexane/EA (7/3) to give the desired product 1 (2.43 g, 36% yield; R f = 0.35) in the form of a very light-yellow clear oil. Its chemical structure was confirmed by 1 H NMR analysis in CDCl 3 ( Figure 2). Toluene was refluxed over CaH2 for 2 h prior to distillation. THF was dried by distillation over sodium-benzophenone when the solution was dark blue. Water content of mPEG45-OH was removed by two times of azeotropic distillation in dry toluene at 175 °C. HMPA and CL were distilled from CaH2 shortly before use. LDA, as a pyrophoric material that is highly flammable, water reactive, toxic, corrosive and known to be carcinogenic and teratogenic, was used according to strict safety guidelines. Deionized water, obtained from a Thermo Scientific Barnstead Nanopure ultra-pure water system, was used for the preparation of all buffer solutions. All other chemicals were used without further purification.

Synthesis of Allyl-Functionalized CL (ACL, 1)
ACL 1 was synthesized according to a previously reported procedure (Scheme 1) [30]. Briefly, 150 mL of freshly distilled dried THF was air-free transferred to a vacuum dried 250-mL reaction flask with a magnetic stirring bar under vigorous nitrogen purging and cooled in a dry ice/acetone bath. Upon reaching a solution temperature of −78 °C, the LDA solution (2.0 M, 25 mL) was carefully added dropwise to the reaction flask. After stirring for a few minutes, a diluted solution of CL (5.00 g) in THF (30 mL) was added slowly over a period of 1 h. This mixture was stirred at −78 °C for another 1 h, and allyl bromide (6 mL) in HMPA (10 mL) was added dropwise. The reaction system was allowed to warm up over 1 h to −45 °C and was kept at this temperature for another 1.5 h. The reaction was quenched by adding a saturated NH4Cl solution to the flask. The reaction mixture was allowed to warm up to room temperature and then washed with saturated NaCl and NH4Cl solutions (3×). The upper organic layer was concentrated and added to cold diethyl ether. The white precipitate was removed by filtration, and the filtrate was dried over magnesium sulfate and concentrated under vacuum. The resulting crude product was purified through a silica gel column chromatography eluted with hexane/EA (7/3) to give the desired product 1 (2.43 g, 36% yield; Rf = 0.35) in the form of a very light-yellow clear oil. Its chemical structure was confirmed by 1 H NMR analysis in CDCl3 ( Figure 2).

Synthesis of PEG-b-(Allyl-Functionalized PCL) (PEG-b-APCL, 2)
PEG-b-APCL 2 was synthesized based on a literature method of ring-opening polymerization (ROP) of ACL 1 using TBD as organocatalyst [31,32]. A 10-mL Schlenk flask with a magnetic stirring bar was flamed dried under vacuum and refilled with nitrogen (3×). Preweighted amount of dried mPEG45-OH macroinitiator (65.69 mg, 33 μmol, 1.0 equiv.) and dry TBD (22.86 mg, 164 μmol, 5.0 equiv.) were dissolved in dry toluene (2.5 mL), and then added to the flask via nitrogen-purged syringe. The solution was stirred for 30 min at room temperature. ACL monomer 1 (500 mg, 3.28 mmol, 100 equiv.) was dissolved in dry toluene (2.5 mL) and added to the flask with an air-free syringe to start ROP at room temperature. GPC and 1 H NMR characterizations were used to monitor the ROP process and monomer conversion, respectively. Once the desired monomer conversion was achieved after 19 h, the reaction was quenched by adding several drops of acetic acid. The polymer was isolated by precipitation in cold diethyl ether (3×) to obtain a beige pasty solid (150 mg, conversion 32%, yield 94%). Figure 3 shows the 500 MHz 1 H NMR spectrum of PEG-b-APCL (2) in CDCl3.

Synthesis of PEG-b-CPCL-70 (3)
In a 10-mL Schlenk flask with a magnetic stirring bar, PEG-b-PCL (2) (150 mg, 705 μmol of allyl group) was dissolved in 5 mL anhydrous chloroform. DEAET (110 mg, 756 μmol, 1.1 equiv. relative to allyl group) and DMPA (36 mg, 141 μmol, 0.2 equiv. relative to allyl group) were added to the reaction flask. The reaction flask was sealed carefully, and the reaction mixture was degassed by five freeze-pump-thaw cycles, followed by irradiation with UV light (λmax = 365 nm) for 40 min. Then the reaction mixture was dialyzed against DCM for 4 days (MW cut-off (MWCO) = 3.5 kDa) and dried under vacuum to obtain PEG-b-CPCL-70 (3, the number 70 indicating mole percent of amine group relative to CL unit) as a white pasty solid (166 mg, 76% isolated yield).   PEG-b-APCL 2 was synthesized based on a literature method of ring-opening polymerization (ROP) of ACL 1 using TBD as organocatalyst [31,32]. A 10-mL Schlenk flask with a magnetic stirring bar was flamed dried under vacuum and refilled with nitrogen (3×). Preweighted amount of dried mPEG 45 -OH macroinitiator (65.69 mg, 33 µmol, 1.0 equiv.) and dry TBD (22.86 mg, 164 µmol, 5.0 equiv.) were dissolved in dry toluene (2.5 mL), and then added to the flask via nitrogen-purged syringe. The solution was stirred for 30 min at room temperature. ACL monomer 1 (500 mg, 3.28 mmol, 100 equiv.) was dissolved in dry toluene (2.5 mL) and added to the flask with an air-free syringe to start ROP at room temperature. GPC and 1 H NMR characterizations were used to monitor the ROP process and monomer conversion, respectively. Once the desired monomer conversion was achieved after 19 h, the reaction was quenched by adding several drops of acetic acid. The polymer was isolated by precipitation in cold diethyl ether (3×) to obtain a beige pasty solid (150 mg, conversion 32%, yield 94%). Figure 3 shows the 500 MHz 1 H NMR spectrum of PEG-b-APCL (2) in CDCl 3 .

Synthesis of PEG-b-(Allyl-Functionalized PCL) (PEG-b-APCL, 2)
PEG-b-APCL 2 was synthesized based on a literature method of ring-opening polymerization (ROP) of ACL 1 using TBD as organocatalyst [31,32]. A 10-mL Schlenk flask with a magnetic stirring bar was flamed dried under vacuum and refilled with nitrogen (3×). Preweighted amount of dried mPEG45-OH macroinitiator (65.69 mg, 33 μmol, 1.0 equiv.) and dry TBD (22.86 mg, 164 μmol, 5.0 equiv.) were dissolved in dry toluene (2.5 mL), and then added to the flask via nitrogen-purged syringe. The solution was stirred for 30 min at room temperature. ACL monomer 1 (500 mg, 3.28 mmol, 100 equiv.) was dissolved in dry toluene (2.5 mL) and added to the flask with an air-free syringe to start ROP at room temperature. GPC and 1 H NMR characterizations were used to monitor the ROP process and monomer conversion, respectively. Once the desired monomer conversion was achieved after 19 h, the reaction was quenched by adding several drops of acetic acid. The polymer was isolated by precipitation in cold diethyl ether (3×) to obtain a beige pasty solid (150 mg, conversion 32%, yield 94%). Figure 3 shows the 500 MHz 1 H NMR spectrum of PEG-b-APCL (2) in CDCl3.

Synthesis of PEG-b-CPCL-70 (3)
In a 10-mL Schlenk flask with a magnetic stirring bar, PEG-b-PCL (2) (150 mg, 705 µmol of allyl group) was dissolved in 5 mL anhydrous chloroform. DEAET (110 mg, 756 µmol, 1.1 equiv. relative to allyl group) and DMPA (36 mg, 141 µmol, 0.2 equiv. relative to allyl group) were added to the reaction flask. The reaction flask was sealed carefully, and the reaction mixture was degassed by five freeze-pump-thaw cycles, followed by irradiation with UV light (λ max = 365 nm) for 40 min. Then the reaction mixture was dialyzed against DCM for 4 days (MW cut-off (MWCO) = 3.5 kDa) and dried under vacuum to obtain PEG-b-CPCL-70 (3, the number 70 indicating mole percent of amine group relative to CL unit) as a white pasty solid (166 mg, 76% isolated yield).

Synthesis of Allyl-Functionalized Poly(ε-caprolactone) (APCL) (4)
Based on a literature method of ring-opening polymerization (ROP) of ACL 1 using TBD as an organocatalyst [31,32], APCL (4) was synthesized. In a 10-mL flamed dried Schlenk flask with a magnetic stirring bar under nitrogen atmosphere, TBD (45 mg, 324 μmol, 3.5 equiv.) was dissolved in dry toluene (5.0 mL), followed by the addition of BnOH (10 mg, 92.6 μmol in a stock solution in dry toluene, 1.0 equiv.). The mixture was then stirred for 30 min at room temperature under nitrogen protection. ACL monomer 1 (1.00 g, 6.50 mmol, 70 equiv.) was dissolved in dry toluene (5.0 mL) and added to the flask with an air-free syringe to start ROP at room temperature. ROP was allowed to continue until it reached desired monomer conversion, as observed by 1 H NMR analysis. After 13.5 h, ROP was quenched by adding several drops of acetic acid. APCL was isolated by precipitation in cold methanol (3×), yielding a light-yellow viscous oil (333 mg, 50% conversion of 1, 67% isolated yield). Figure 5 illustrates the 500 MHz 1 H NMR spectrum of APCL (4) in DMSO-d6.

Synthesis of Allyl-Functionalized Poly(ε-caprolactone) (APCL) (4)
Based on a literature method of ring-opening polymerization (ROP) of ACL 1 using TBD as an organocatalyst [31,32], APCL (4) was synthesized. In a 10-mL flamed dried Schlenk flask with a magnetic stirring bar under nitrogen atmosphere, TBD (45 mg, 324 µmol, 3.5 equiv.) was dissolved in dry toluene (5.0 mL), followed by the addition of BnOH (10 mg, 92.6 µmol in a stock solution in dry toluene, 1.0 equiv.). The mixture was then stirred for 30 min at room temperature under nitrogen protection. ACL monomer 1 (1.00 g, 6.50 mmol, 70 equiv.) was dissolved in dry toluene (5.0 mL) and added to the flask with an air-free syringe to start ROP at room temperature. ROP was allowed to continue until it reached desired monomer conversion, as observed by 1 H NMR analysis. After 13.5 h, ROP was quenched by adding several drops of acetic acid. APCL was isolated by precipitation in cold methanol (3×), yielding a light-yellow viscous oil (333 mg, 50% conversion of 1, 67% isolated yield). Figure 5 illustrates the 500 MHz 1 H NMR spectrum of APCL (4) in DMSO-d 6 .

Synthesis of Allyl-Functionalized Poly(ε-caprolactone) (APCL) (4)
Based on a literature method of ring-opening polymerization (ROP) of ACL 1 using TBD as an organocatalyst [31,32], APCL (4) was synthesized. In a 10-mL flamed dried Schlenk flask with a magnetic stirring bar under nitrogen atmosphere, TBD (45 mg, 324 μmol, 3.5 equiv.) was dissolved in dry toluene (5.0 mL), followed by the addition of BnOH (10 mg, 92.6 μmol in a stock solution in dry toluene, 1.0 equiv.). The mixture was then stirred for 30 min at room temperature under nitrogen protection. ACL monomer 1 (1.00 g, 6.50 mmol, 70 equiv.) was dissolved in dry toluene (5.0 mL) and added to the flask with an air-free syringe to start ROP at room temperature. ROP was allowed to continue until it reached desired monomer conversion, as observed by 1 H NMR analysis. After 13.5 h, ROP was quenched by adding several drops of acetic acid. APCL was isolated by precipitation in cold methanol (3×), yielding a light-yellow viscous oil (333 mg, 50% conversion of 1, 67% isolated yield). Figure 5 illustrates the 500 MHz 1 H NMR spectrum of APCL (4) in DMSO-d6.
Materials 2020, 13, x FOR PEER REVIEW 7 of 17 yellow and brittle white hygroscopic solids, respectively. Figure 6 illustrates the 500 MHz 1 H NMR spectrum of CPCL-50 in DMSO-d6, which is similar to the spectrum of CPCL-95 (not shown).

Study of Hydrolytic Stability of PEG-b-CPCL-70 (3)
Two PBS buffer solutions of pH 5.5 and 7.4 at a concentration of 25 mM were prepared. PEG-b-CPCL-70 was dissolved in prepared buffer medium at a concentration of 1 mg/mL. The solutions were then transferred to separate sealed vials and incubated at 37 °C with mild shaking. At specific time intervals, aliquots from each vial were taken and completely dried under vacuum to remove any moisture. The resulting solid was dissolved in DMF (containing 0.01 M LiBr) and analyzed with GPC to assess hydrolytic stability.

Preparation of Polymer:pDNA Nanocomplexes
Nanocomplexes were formed based on spontaneous electrostatic interactions between PEG-b-CPCL-70 and pCMV-EGFP pDNA at various polymer:pDNA mass ratios. The pDNAs were produced by an Escherichia coli bacterial host (GeneHogs, Invitrogen), with an enhanced green fluorescent protein reporter gene, driven by a cytomegalovirus promoter within plasmids (pCMV-EGFP, Addgene). A stock aqueous solution of polymer was prepared at a concentration of 4 mg/mL in ultrapure water. For each polymer:pDNA nanocomplex mass ratio, a specific amount of polymer solution was mixed with a pDNA solution, gently vortexed, and incubated for 30 min to ensure the formation of polymer:pDNA nanocomplexes. For example, for the preparation of nanocomplex at 200:1 of PEG-b-CPCL-70:pDNA mass ratio, 400 μL of polymer stock solution (4 mg/mL) was mixed with pCMV-EGFP pDNA (with a final concentration of 8 μg/mL) to a final volume of 1 mL.

N/P Ratio Calculation
N/P ratio denotes the molecular ratio of cationic amine groups (N) in the PEG-b-CPCL-70 to anionic phosphate groups (P) in pDNA [33]. The mass per mole for one anionic charge (P) of pDNA is 330 g/mol [34], whereas the mass per mole for one cationic charge (N) of PEG-b-CPCL-70 is 465 g/mol. The value of 465 g/mol was obtained by dividing Mn NMR of PEG-b-CPCL-70 with the number of cationic amine groups (N) of the polymer. Therefore, in a PEG-b-CPCL-70:pDNA nanocomplex, the N/P ratio was calculated as: This equation was used to convert PEG-b-CPCL-70:pDNA mass ratios to N/P ratios (N/P ratios = 0.71 mass ratios).

Study of Hydrolytic Stability of PEG-b-CPCL-70 (3)
Two PBS buffer solutions of pH 5.5 and 7.4 at a concentration of 25 mM were prepared. PEG-b-CPCL-70 was dissolved in prepared buffer medium at a concentration of 1 mg/mL. The solutions were then transferred to separate sealed vials and incubated at 37 • C with mild shaking. At specific time intervals, aliquots from each vial were taken and completely dried under vacuum to remove any moisture. The resulting solid was dissolved in DMF (containing 0.01 M LiBr) and analyzed with GPC to assess hydrolytic stability.

Preparation of Polymer:pDNA Nanocomplexes
Nanocomplexes were formed based on spontaneous electrostatic interactions between PEG-b-CPCL-70 and pCMV-EGFP pDNA at various polymer:pDNA mass ratios. The pDNAs were produced by an Escherichia coli bacterial host (GeneHogs, Invitrogen), with an enhanced green fluorescent protein reporter gene, driven by a cytomegalovirus promoter within plasmids (pCMV-EGFP, Addgene). A stock aqueous solution of polymer was prepared at a concentration of 4 mg/mL in ultrapure water. For each polymer:pDNA nanocomplex mass ratio, a specific amount of polymer solution was mixed with a pDNA solution, gently vortexed, and incubated for 30 min to ensure the formation of polymer:pDNA nanocomplexes. For example, for the preparation of nanocomplex at 200:1 of PEG-b-CPCL-70:pDNA mass ratio, 400 µL of polymer stock solution (4 mg/mL) was mixed with pCMV-EGFP pDNA (with a final concentration of 8 µg/mL) to a final volume of 1 mL.

N/P Ratio Calculation
N/P ratio denotes the molecular ratio of cationic amine groups (N) in the PEG-b-CPCL-70 to anionic phosphate groups (P) in pDNA [33]. The mass per mole for one anionic charge (P) of pDNA is 330 g/mol [34], whereas the mass per mole for one cationic charge (N) of PEG-b-CPCL-70 is 465 g/mol.
This equation was used to convert PEG-b-CPCL-70:pDNA mass ratios to N/P ratios (N/P ratios = 0.71 mass ratios).

pDNA Gel-shift Assay
The polymer:pDNA nanocomplexes were tested with a gel electrophoresis assay. Samples of nanocomplexes were loaded into a 1% agarose gel with ethidium bromide staining and electrophoresed at 100 V for 30 min. Gels were visualized under UV after electrophoresis to evaluate the pDNA shift at various polymer:pDNA mass ratios for nanocomplex formation.

pDNA Release by Polyanionic Heparin
The binding of pDNA to the polymeric domain within the nanocomplex was examined using polyanionic heparin to release the pDNA [35]. Nanocomplexes were prepared at a polymer:pDNA mass ratio of 200:1 to guarantee the complete condensation of pDNA in the cationic polymer matrix, followed by incubation at 37 • C for 1 h. The samples with various nanocomplex:heparin mass ratios were run on an agarose gel electrophoresis system as described earlier.

Serum Stability of Polymer:pDNA Nanocomplexes
The stability of pDNA in PEG-b-CPCL-70:pDNA nanocomplexes against fetal bovine serum (FBS, Atlanta Biologicals) was studied by agarose gel electrophoresis [36]. A 300 µL solution of polymer:pDNA nanocomplexes at a 200:1 mass ratio was incubated with 10% FBS for 24 h. Aliquots of 25 µL at different time points were taken and frozen immediately. Prior to agarose gel electrophoresis, 4 µL (1 mg/mL) of polyanionic heparin solution was added to each aliquot to ensure the release of pDNA from nanocomplexes. Intact pDNA species were then assessed by comparing DNA fragmentation patterns.

Synthesis and Characterization of PEG-b-CPCL-70 (3) and CPCLs (5)
Aliphatic polyesters are among the most widely studied synthetic polymers for biomedical applications due to their remarkable biodegradability. In our previous studies, we investigated the delivery of genetic material, such as pDNA and siRNA, using CPLAs [25][26][27]. It was found that these PLA-based materials have promising transfection efficiency, but they suffer from fast hydrolytic degradation (i.e., they degrade in aqueous solutions within a week), which limits their practical application [25,27]. Besides PLA, PCL composed of hexanoate repeating units is also a predominant aliphatic polyester and has been utilized extensively in biomedical applications. PCL has a lower density of ester groups on its backbone, and, therefore, offers slower degradation under biologically relevant conditions in comparison to PLA. The degradation of PCL produces 6-hydroxyhexanoic acid as the eventual small-molecule degradation residue, which is a natural human body metabolite. Generally, PCL can biodegrade over a longer time span from several months to several years, depending on MW, degree of crystallinity, and the condition of degradation [37]. Moreover, relative to PLA, PCL provides a more hydrophobic domain, which is less accessible to water due to the presence of five hydrophobic methylene moieties on each repeating unit of PCL. Therefore, in this study, we investigated tertiary amine-based CPCL polymers for pDNA delivery. Specifically, PEGylated CPCL, i.e., PEG-b-CPCL, was the polymer of interest in this investigation because of the merit of PEGylated synthetic vectors, as revealed in our previous work [25]. Non-PEGylated CPCLs were used along with PEGylated CPCLs in buffer solubility tests.
Because polymer synthesis for advanced applications such as drug/gene delivery requires the control over the polymer MW, MW dispersity (Đ), and functional groups, synthetic routes of these CPCL-based polymers were designed with two key steps: (1) the living ROP of ACL 1 to obtain APCL-based precursor polymers, and (2) the subsequent post-polymerization transformation of allyl groups to tertiary amine groups via thiol-ene click functionalization. ACL 1 was prepared following an approach from the literature (Scheme 1) [30], with chemical structure verified by 1 H NMR analysis in CDCl 3 (Figure 2). Scheme 2 illustrates the synthesis of PEGylated and non-PEGylated CPCLs. In the living ROP of 1, TBD was used as the organocatalyst because it has relatively high catalytic efficacy that can result in dual activation of both monomer and alcoholic initiator [31], leading to the ROP process of CL monomer in a rapid and well-controlled manner (with a linear relationship between conversion and MW). The preparation of PEG-b-APCL, i.e., the diblock precursor of PEG-b-CPCL, was carried out in toluene at room temperature for 19 h by using mPEG 45 (Figure 4). GPC analysis showed a Mn GPC of 9.6 kDa with a Đ GPC of 1.15 for 3. It should be noted that the low Đ of 3 is preferred for gene delivery applications, because polymers with high Đ values may potentially lead to nanocomplexes with broad size distributions and consequently less controlled biophysical and biomedical properties. GPC curves of mPEG45-OH macroinitiator, 2, and 3 are shown in Figure 7a. It should be noted that the low Đ of 3 is preferred for gene delivery applications, because polymers with high Đ values may potentially lead to nanocomplexes with broad size distributions and consequently less controlled biophysical and biomedical properties. GPC curves of mPEG 45 -OH macroinitiator, 2, and 3 are shown in Figure 7a.

Hydrolytic Stability of PEG-b-CPCL-70 (3)
As compared with PLA, PCL as a biodegradable polyester has very slow hydrolytic degradation, and literature reports that it takes more than a year for either bulk or nanostructured PCL to start showing major degradations under aqueous environments at 37 • C [38], although the degradation of PCL becomes faster at elevated temperatures [39,40]. The hydrolytic stability of PCL relative to PLA is among the key considerations for us to design CPCL-based polymers for gene delivery applications. Before performing a hydrolytic stability study for CPCL-based polymers, their aqueous solubility (Table 1) was probed at the concentration of 1.0 mg/mL in 25 mM PBS buffer at 37 • C and physiologically relevant pH values of 7.4 (representative pH for human blood) and 5.5 (representative pH for endosomes). PEG 45 -b-CPCL 32 -70 showed aqueous solubility at both pHs. On the other hand, CPCL 35 -50, and CPCL 35 -95 were only soluble at pH 5.5; they formed cloudy dispersions at pH 7.4 with the assistance of sonication, but polymers precipitated a few days later. The pH-dependent solubility of CPCLs can be ascribed to the protonation of the amine group at lower pH that favors a dissolving process. The incorporation of the hydrophilic PEG block with CPCL leads to higher and less pH-dependent solubility of the resulting diblock copolymer relative to CPCLs. Therefore, the subsequent hydrolytic stability study was focused on PEG 45 -b-CPCL 32 -70 (3).   (Figure 7b,c). GPC results showed similar hydrolytic stability of 3 at both pH 5.5 and 7.4. Throughout the period, the polymer peak maintained its original position with gradually appearing shoulders. The shoulder at the low-MW side was less significant, indicating a quite slow hydrolytic degradation. The shoulder at the high-MW side became more noticeable after the first week of incubation, presumably because of the minor occurrence of intermolecular linking reactions of CPCL blocks via the remaining allyl groups. In contrast to the relatively fast degradation of CPLAs [25], the remarkable hydrolytic stability of 3 can be attributed to its significantly lower ester group density on the more hydrophobic aliphatic polyester backbone.

Nanocomplex Formation and Characterization
Gel electrophoresis is a direct approach to assess the stability of pDNA in a gene delivery formulation. In order to evaluate the appropriate polymer:pDNA mass ratio for the nanocomplex formation (N/P = 0.71 is equivalent to polymer:pDNA mass ratio of 1:1), agarose gel electrophoresis was implemented to visualize how much PEG 45 -b-CPCL 32 -70 is needed to complex with pCMV-EGFP pDNA (Figure 9). At a 1:1 mass ratio of polymer:pDNA (lane 8), most of the pDNA was not complexed and showed migration similar to naked pDNA (lane 9). At mass ratios of 10:1 (lane 7) and 20:1 (lane 6), the migration of pDNA in the electric field was still noticeable, evidently slower than naked pDNA, indicating that free pDNA was absent, but the electrostatic interaction of pDNA with polymer was relatively weak. It was observed that at a polymer:pDNA mass ratio of 50:1 (lane 5), migration of pDNA was significantly retarded, indicating a high level of complexation of PEG 45 -b-CPCL 32 -70 with pCMV-EGFP pDNA. Further increase in the mass ratio (100:1 or higher; lanes 1-4) resulted in an even slower migration of pDNA, indicating the stronger electrostatic interaction of pDNA with the polymer. The surface charge and hydrodynamic size of the nanocomplexes in water were further investigated by dynamic light scattering (DLS) analysis. As shown in Figure 10, the number-average hydrodynamic diameter (Dh,N) of nanocomplexes increased with the increase in polymer:pDNA mass ratio in the range of 20:1 (Dh,N = 118 ± 5 nm) to 200:1 (Dh,N = 240 ± 4 nm). Such tunable sizes of the nanocomplexes may be helpful to optimize their gene delivery efficacy because gene transfer studies using parenchymal liver cells as the model cell line suggested that the diameter of sinusoidal fenestrae exerts a size limit for effective vectors [41,42]. Because PEG45-b-CPCL32-70 is positively charged, zeta potential (ζ) of nanocomplexes also increased with the increase in polymer:pDNA mass ratio in the range of 20:1 (ζ = 9 ± 1 mV) to 200:1 (ζ = 26 ± 2 mV). The significant ζ values observed for nanocomplexes at high polymer:pDNA mass ratios can promote their colloidal stability, which is important for gene delivery application. The morphology and size of nanocomplexes were investigated with transmission electron microscopy according to our previously published sample preparation method (TEM; Figure 11) [43]. The polymer:pDNA ratio of 200:1 for nanocomplexes was selected for TEM characterization, and the corresponding TEM images showed their rough spherical morphologies, with a number-average The surface charge and hydrodynamic size of the nanocomplexes in water were further investigated by dynamic light scattering (DLS) analysis. As shown in Figure 10, the number-average hydrodynamic diameter (D h,N ) of nanocomplexes increased with the increase in polymer:pDNA mass ratio in the range of 20:1 (D h,N = 118 ± 5 nm) to 200:1 (D h,N = 240 ± 4 nm). Such tunable sizes of the nanocomplexes may be helpful to optimize their gene delivery efficacy because gene transfer studies using parenchymal liver cells as the model cell line suggested that the diameter of sinusoidal fenestrae exerts a size limit for effective vectors [41,42]. Because PEG 45 -b-CPCL 32 -70 is positively charged, zeta potential (ζ) of nanocomplexes also increased with the increase in polymer:pDNA mass ratio in the range of 20:1 (ζ = 9 ± 1 mV) to 200:1 (ζ = 26 ± 2 mV). The significant ζ values observed for nanocomplexes at high polymer:pDNA mass ratios can promote their colloidal stability, which is important for gene delivery application. The surface charge and hydrodynamic size of the nanocomplexes in water were further investigated by dynamic light scattering (DLS) analysis. As shown in Figure 10, the number-average hydrodynamic diameter (Dh,N) of nanocomplexes increased with the increase in polymer:pDNA mass ratio in the range of 20:1 (Dh,N = 118 ± 5 nm) to 200:1 (Dh,N = 240 ± 4 nm). Such tunable sizes of the nanocomplexes may be helpful to optimize their gene delivery efficacy because gene transfer studies using parenchymal liver cells as the model cell line suggested that the diameter of sinusoidal fenestrae exerts a size limit for effective vectors [41,42]. Because PEG45-b-CPCL32-70 is positively charged, zeta potential (ζ) of nanocomplexes also increased with the increase in polymer:pDNA mass ratio in the range of 20:1 (ζ = 9 ± 1 mV) to 200:1 (ζ = 26 ± 2 mV). The significant ζ values observed for nanocomplexes at high polymer:pDNA mass ratios can promote their colloidal stability, which is important for gene delivery application. The morphology and size of nanocomplexes were investigated with transmission electron microscopy according to our previously published sample preparation method (TEM; Figure 11) [43]. The polymer:pDNA ratio of 200:1 for nanocomplexes was selected for TEM characterization, and the corresponding TEM images showed their rough spherical morphologies, with a number-average The morphology and size of nanocomplexes were investigated with transmission electron microscopy according to our previously published sample preparation method (TEM; Figure 11) [43]. The polymer:pDNA ratio of 200:1 for nanocomplexes was selected for TEM characterization, and the corresponding TEM images showed their rough spherical morphologies, with a number-average surface diameter of 135 ± 17 nm. TEM provided a smaller average size and narrower size distribution of nanocomplexes than DLS for the same nanocomplexes ( Figure 12) because the nanocomplexes dried and considerably shrank on the TEM grid while DLS gave hydrodynamic size distribution. surface diameter of 135 ± 17 nm. TEM provided a smaller average size and narrower size distribution of nanocomplexes than DLS for the same nanocomplexes ( Figure 12) because the nanocomplexes dried and considerably shrank on the TEM grid while DLS gave hydrodynamic size distribution.

pDNA Release and Serum Stability of Nanocomplexes
It is important to assess whether the nanocomplexes can provide protection for pDNA and also allow the release of pDNA in biological conditions. Because direct observation of these phenomena under cellular environments is more difficult, the release of pDNA from the nanocomplexes in aqueous solutions in the presence of heparin (a model anionic polyelectrolyte biopolymer) and the serum stability of the nanocomplexes were probed. Agarose gel electrophoresis was utilized in pDNA analysis and nanocomplexes prepared at polymer:pDNA mass ratio of 200:1 (N/P = 142) were employed in each study.
Heparin, as an anionic polyelectrolyte, is able to break the nanocomplex formed from PEG-b-CPCL-70 and pDNA. In this study, various amounts of heparin were used to compete with PEG-b-CPCL-70 until the pDNA was released from the nanocomplex. At a fixed polymer:pDNA mass ratio of 200:1, which ensured nanocomplex formation, heparin solutions were added gradually, and the mixed solutions were obtained with vortex stirring. The solutions were then incubated at 37 °C for 1 h, followed by running gel electrophoresis to evaluate the unbinding behavior. Figure 13 illustrates the UV visualized image of gel electrophoresis stained by ethidium bromide. It is observed that, with the increase in the heparin mass, the pDNA started to unbind form the nanocomplex matrix. This can be presumably attributed to electrostatic interaction between heparin and PEG-b-CPCL-70, which are negative and positive polyelectrolytes, respectively. An incremental increase in the amount of heparin provided more negative charge such that it could overcompensate the existing positive surface diameter of 135 ± 17 nm. TEM provided a smaller average size and narrower size distribution of nanocomplexes than DLS for the same nanocomplexes ( Figure 12) because the nanocomplexes dried and considerably shrank on the TEM grid while DLS gave hydrodynamic size distribution.

pDNA Release and Serum Stability of Nanocomplexes
It is important to assess whether the nanocomplexes can provide protection for pDNA and also allow the release of pDNA in biological conditions. Because direct observation of these phenomena under cellular environments is more difficult, the release of pDNA from the nanocomplexes in aqueous solutions in the presence of heparin (a model anionic polyelectrolyte biopolymer) and the serum stability of the nanocomplexes were probed. Agarose gel electrophoresis was utilized in pDNA analysis and nanocomplexes prepared at polymer:pDNA mass ratio of 200:1 (N/P = 142) were employed in each study.
Heparin, as an anionic polyelectrolyte, is able to break the nanocomplex formed from PEG-b-CPCL-70 and pDNA. In this study, various amounts of heparin were used to compete with PEG-b-CPCL-70 until the pDNA was released from the nanocomplex. At a fixed polymer:pDNA mass ratio of 200:1, which ensured nanocomplex formation, heparin solutions were added gradually, and the mixed solutions were obtained with vortex stirring. The solutions were then incubated at 37 °C for 1 h, followed by running gel electrophoresis to evaluate the unbinding behavior. Figure 13 illustrates the UV visualized image of gel electrophoresis stained by ethidium bromide. It is observed that, with the increase in the heparin mass, the pDNA started to unbind form the nanocomplex matrix. This can be presumably attributed to electrostatic interaction between heparin and PEG-b-CPCL-70, which are negative and positive polyelectrolytes, respectively. An incremental increase in the amount of heparin provided more negative charge such that it could overcompensate the existing positive

pDNA Release and Serum Stability of Nanocomplexes
It is important to assess whether the nanocomplexes can provide protection for pDNA and also allow the release of pDNA in biological conditions. Because direct observation of these phenomena under cellular environments is more difficult, the release of pDNA from the nanocomplexes in aqueous solutions in the presence of heparin (a model anionic polyelectrolyte biopolymer) and the serum stability of the nanocomplexes were probed. Agarose gel electrophoresis was utilized in pDNA analysis and nanocomplexes prepared at polymer:pDNA mass ratio of 200:1 (N/P = 142) were employed in each study.
Heparin, as an anionic polyelectrolyte, is able to break the nanocomplex formed from PEG-b-CPCL-70 and pDNA. In this study, various amounts of heparin were used to compete with PEG-b-CPCL-70 until the pDNA was released from the nanocomplex. At a fixed polymer:pDNA mass ratio of 200:1, which ensured nanocomplex formation, heparin solutions were added gradually, and the mixed solutions were obtained with vortex stirring. The solutions were then incubated at 37 • C for 1 h, followed by running gel electrophoresis to evaluate the unbinding behavior. Figure 13 illustrates the UV visualized image of gel electrophoresis stained by ethidium bromide. It is observed that, with the increase in the heparin mass, the pDNA started to unbind form the nanocomplex matrix. This can be presumably attributed to electrostatic interaction between heparin and PEG-b-CPCL-70, which are negative and positive polyelectrolytes, respectively. An incremental increase in the amount of heparin provided more negative charge such that it could overcompensate the existing positive charge in the nanocomplex. Consequently, pDNA species were not bound to any cationic domain and able to be released completely. From lane 3 to 9, pDNA species were gradually able to drift down the in the electric field until in lane 10, and they were completely released as they gave the same spot lines as naked pDNA in lane 11. Although heparin-triggered pDNA release is not cell-realistic and there are many kinds of anionic species under cellular environments, the above result demonstrates that pDNA can be released from the nanocomplexes via electrostatic interactions.
Materials 2020, 13, x FOR PEER REVIEW 14 of 17 charge in the nanocomplex. Consequently, pDNA species were not bound to any cationic domain and able to be released completely. From lane 3 to 9, pDNA species were gradually able to drift down the in the electric field until in lane 10, and they were completely released as they gave the same spot lines as naked pDNA in lane 11. Although heparin-triggered pDNA release is not cell-realistic and there are many kinds of anionic species under cellular environments, the above result demonstrates that pDNA can be released from the nanocomplexes via electrostatic interactions. Serum endonuclease is capable of fragmenting unprotected pDNA during in vivo transport. One of the most important features of suitable polymeric gene delivery systems is to protect pDNA formulations in the serum environment. Unprotected pDNA can undergo a fast break down in time scales of less than an hour due to serum endonuclease [36]. The shielding feature of the PEG-b-CPCL-70 polymeric gene delivery system for pDNA against FBS was investigated directly with agarose gel electrophoresis ( Figure 14). Accordingly, 300 μL of polymer:pDNA nanocomplexes at a 200:1 mass ratio (N/P = 142) was combined with 10% FBS and incubated at 37 °C for 24 h. At various time points, aliquots of 25 μL were taken and frozen immediately to stop the serum degradation activity. Prior to agarose gel electrophoresis, the samples were treated with 4 μL (1 mg/mL) polyanionic heparin to enable pDNA to migrate in gel electrophoresis. Intact pDNA species demonstrate similar migration and ethidium staining (lanes 2 to 10 in Figure 14) compared to the pDNA in lane 1 without incubation. Whereas, after 24 h of incubation, the pDNA in lane 11 was almost entirely degraded to smaller oligonucleotides.  Serum endonuclease is capable of fragmenting unprotected pDNA during in vivo transport. One of the most important features of suitable polymeric gene delivery systems is to protect pDNA formulations in the serum environment. Unprotected pDNA can undergo a fast break down in time scales of less than an hour due to serum endonuclease [36]. The shielding feature of the PEG-b-CPCL-70 polymeric gene delivery system for pDNA against FBS was investigated directly with agarose gel electrophoresis ( Figure 14). Accordingly, 300 µL of polymer:pDNA nanocomplexes at a 200:1 mass ratio (N/P = 142) was combined with 10% FBS and incubated at 37 • C for 24 h. At various time points, aliquots of 25 µL were taken and frozen immediately to stop the serum degradation activity. Prior to agarose gel electrophoresis, the samples were treated with 4 µL (1 mg/mL) polyanionic heparin to enable pDNA to migrate in gel electrophoresis. Intact pDNA species demonstrate similar migration and ethidium staining (lanes 2 to 10 in Figure 14) compared to the pDNA in lane 1 without incubation. Whereas, after 24 h of incubation, the pDNA in lane 11 was almost entirely degraded to smaller oligonucleotides.

Conclusions
Materials 2020, 13, x FOR PEER REVIEW 14 of 17 charge in the nanocomplex. Consequently, pDNA species were not bound to any cationic domain and able to be released completely. From lane 3 to 9, pDNA species were gradually able to drift down the in the electric field until in lane 10, and they were completely released as they gave the same spot lines as naked pDNA in lane 11. Although heparin-triggered pDNA release is not cell-realistic and there are many kinds of anionic species under cellular environments, the above result demonstrates that pDNA can be released from the nanocomplexes via electrostatic interactions. Serum endonuclease is capable of fragmenting unprotected pDNA during in vivo transport. One of the most important features of suitable polymeric gene delivery systems is to protect pDNA formulations in the serum environment. Unprotected pDNA can undergo a fast break down in time scales of less than an hour due to serum endonuclease [36]. The shielding feature of the PEG-b-CPCL-70 polymeric gene delivery system for pDNA against FBS was investigated directly with agarose gel electrophoresis ( Figure 14). Accordingly, 300 μL of polymer:pDNA nanocomplexes at a 200:1 mass ratio (N/P = 142) was combined with 10% FBS and incubated at 37 °C for 24 h. At various time points, aliquots of 25 μL were taken and frozen immediately to stop the serum degradation activity. Prior to agarose gel electrophoresis, the samples were treated with 4 μL (1 mg/mL) polyanionic heparin to enable pDNA to migrate in gel electrophoresis. Intact pDNA species demonstrate similar migration and ethidium staining (lanes 2 to 10 in Figure 14) compared to the pDNA in lane 1 without incubation. Whereas, after 24 h of incubation, the pDNA in lane 11 was almost entirely degraded to smaller oligonucleotides.

Conclusions
In summary, well-defined cationic PEG-b-CPCL was studied as a potential PEGylated non-viral vector for pDNA delivery. It was synthesized by ROP of ACL using PEO 45 -OH macroinitiator, followed by thiol-ene functionalization of the resulting diblock PEG-b-APCL. Its chemical structure was confirmed by 1 H NMR and GPC analysis. PEG-b-CPCL exhibited significant hydrolytic stability and did not show a major occurrence of hydrolytic degradation in incubation for 12 weeks. Relative to CPCLs with pH-dependent solubility, PEG-b-CPCL can provide solubility over a wide range of pH. It can complex with pDNA to form positively surface-charged spherical nanoparticles, with both hydrodynamic size and surface charge increasing with the increase in the polymer:pDNA ratio. The nanocomplexes can protect pDNA and also allow the release of pDNA. PEG-b-CPCL possesses a remarkable combination of biomedically relevant properties (i.e., significant hydrolytic stability, biodegradability, and complexation ability with pDNA). The above results encourage further studies to investigate PEG-b-CPCL as a promising synthetic vector for gene delivery applications.