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Gene Transfection Mediated by Catiomers Requires Free Highly Charged Polymer Chains to Overcome Intracellular Barriers Lindomar Jose Calumby Albuquerque, Carlos E. de Castro, Karin A. Riske, Maria Cristina Carlan da Silva, Paulo I. R. Muraro, Vanessa Schmidt Giacomelli, Cristiano Giacomelli, and Fernando Carlos Carlos Giacomelli Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on April 30, 2017

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Gene Transfection Mediated by Catiomers Requires Free Highly Charged Polymer Chains to Overcome Intracellular Barriers

Lindomar J. C. Albuquerque,† Carlos E. de Castro,† Karin A. Riske,‡ Maria C. Carlan da Silva,† Paulo I. R. Muraro,# Vanessa Schmidt,# Cristiano Giacomelli,# and Fernando C. Giacomelli†,*

†

Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, Brazil. ‡

#

Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil.

Departamento de Química, Universidade Federal de Santa Maria, Santa Maria, Brazil.

*Corresponding Author:

Fernando Carlos Giacomelli e-mail: [email protected]

ACS Paragon Plus Environment

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Abstract The prospective use of the block copolymers PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) as non-viral gene vectors was evaluated. The polymers are able to properly condense DNA into nano-sized particles (RH ~ 75 nm) which are marginally cytotoxic and can be uptaken by cells. However, the GFP expression assays evidenced that DNA delivery is essentially negligible meaning that intracellular trafficking hampers efficient gene release. Subsequently we demonstrate that cellular uptake and particularly the quantity of GFP-positive cells are substantially enhanced when the block copolymer polyplexes are produced and further supplemented by BPEI chains (branched polyethyleneimine). The DLS/ELS/ITC data suggest that such a strategy allows the adsorption of BPEI onto the surface of the polyplexes, and this phenomenon is responsible for increasing the size and surface charge of the assemblies. Nevertheless, most of the BPEI chains remain freely diffusing in the systems. The biological assays confirmed that cellular uptake is enhanced in the presence of BPEI and principally, the free highly charged polymer chains plays the central role in intracellular trafficking and gene transfection. These investigations pointed out that the transfection efficiency vs. cytotoxicity issue can be balanced by a mixture of BPEI and less cytotoxic agents such as for instance the proposed block copolymers.


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1. Introduction The gene therapy relies on a groundbreaking approach to face generally hereditary diseases where genetic material has to be replaced within the unhealthy cells. Therefore, the DNA is the therapeutic entity. The biological macromolecule, however, is not able to enter the cells by itself because the anionic phosphate backbone is electrostatically repelled by the negatively charged plasma membrane. Thus, the development of DNA cargo delivery systems is required.1 Accordingly, viral vectors have been previously demonstrated to provide high gene transfer and expression efficiency mostly related to their ability to overcome extra and intracellular barriers and to escape the defense mechanisms of the body. On the other hand, despite almost half a century of research, only very limited success has been achieved by using viral vectors particularly because viruses are able to stimulate immune responses, mutagenesis and oncogenesis, besides being potential pathogenic.2–5 In this framework, the development of synthetic gene carriers has emerged as an alternative due to essentially safety issues6,7 where the majority of the proposed cargo agents are based on the complexation of catiomers and DNA via electrostatic interactions.8,9 Nevertheless, although the catiomer/DNA polyplexes science has also been born decades ago, this class of prospective gene carriers still suffers of fundamental drawbacks including low transfection efficiency and significant cytotoxicity. Indeed, even the best currently known vectors remain much less efficient than viruses. Consequently, the vector properties must be certainly optimized and this is still the main challenge of the field. Truly, the intracellular trafficking of polyplexes and particularly the DNA release mechanism remain heavily debated.8,10 Nano-sized polyplexes are usually accepted to be uptaken by cells via endocytosis, however, from this point forward there is no consensus regarding the cellular pathways. Such knowledge is still in its rising phase and many aspects have to be clarified namely:


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endosomal escape mechanism, DNA release and DNA nuclei internalization.11–13 The viruses have evolved mechanisms to overcome these barriers whereas the main weakness of the synthetic vectors is claimed to be the escape from the endocytic vesicles. The “proton sponge” hypothesis was previously proposed to explain the high transfection activity particularly for polyethyleneimine (PEI) polyplexes. The mechanism relies on the capability of polyamines to capture protons provided by the VATPase mediated H+ influx thereby preventing the acidification of the vesicles. The increased concentration of positive charges within the endosomes results in osmotic swelling and ultimately vesicle rupture.13–15 Nevertheless, many catiomers with buffering capacity have shown very low transfection activity.16,17 Along these lines, it has been recently proposed that it is indeed the uncomplexed fraction of polycations the main responsible in promoting transgene expression and not the polyplexes themselves (complexed chains).15-18 The free chains promote gene transfection by acting in different steps (with different extents) of the intracellular trafficking, namely: (i) reducing the lysosomal entrapment of polyplexes, (ii) assisting the cytosol-to-nucleus transportation of pDNA, (iii) enhancing pDNA-to-mRNA transcription efficiency and, (iv) smoothing the nucleus-to-cytosol translocation of mRNA.22 Herein, we investigated the ability of the block copolymers PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-coDPA47) to provide gene transfection. They were previously demonstrated to properly condense DNA into particles with desirable size for cellular uptake via endocytic pathways (RH ~ 65-85 nm). The PDEA units are sufficiently protonated at physiological pH (pKa ~ 7.5) and participate in the ion complex formation with DNA. The higher degree of protonation of PDEA (See Fig. S1) or PDPA (pKa ~ 6.8) at lower pH was supposed to allow endosomal disruption via pH buffering effects at the acidic late endosomes (pH ~ 5) thereby enhancing transfection levels. The PEO or POEGMA


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hydrophilic segments are required for serum stability issues. Nevertheless, we demonstrated that even by using the dual environmentally-responsive block copolymer POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) it was not possible to reach gene expression efficiently. On the other hand, the scenario changes considerably when branched PEI chains (BPEI) are added to the systems after the complete DNA condensation promoted by the amphiphilic block copolymers. Accordingly, the reported results support that, indeed, a free fraction of highly charged polymer chains (but not the polyplexes themselves) mediates transgene expression. We thus make evident that DNA can be carried by using block copolymers of low cytotoxicity and the further addition of a more cytotoxic agent (BPEI was herein chosen) enable endosomal release. This means that successful gene expression might be obtained using formulations of overall lower cytotoxicity towards more efficient gene delivery systems.

2. Experimental Section 2.1. Materials and Chemicals The block copolymers were synthesized by either atom transfer radical (ATRP) or reversible addition-fragmentation chain transfer (RAFT) polymerization techniques using

already

well-established

protocols.

Poly(ethylene

oxide)113-b-poly[2-

(diethylamino)ethyl methacrylate]50 (PEO113-b-PDEA50, Mn = 14500 g.mol-1, Mw/Mn = 1.20) was synthesized following the procedure described by Liu et al.23 with adaptations as described in details elsewhere.24 Poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly[oligo(ethylene (diethylamino)ethyl

glycol)methyl

ether

methacrylate10-co-2-

methacrylate47-co-2-(diisopropylamino)ethyl

methacrylate47]

(POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47), Mn = 59000 g.mol-1, Mw/Mn = 1.28) was synthesized by RAFT polymerization in toluene employing a one-pot/two-step


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approach

involving

the

sequential

addition

of

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monomers

and

4-cyano-4-

[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid chain transfer agent, as adapted from a recent study by our group.25 Branched polyethyleneimine (BPEI, Mn = 25000 g.mol-1, Mw/Mn = 2.50) was purchased from Sigma Aldrich. The molecular structure of the polymer samples is shown in Figure 1. Details of synthesis procedures along to NMR spectra and GPC chromatograms of the mentioned block copolymers were recently published.26 Calf Thymus DNA was purchased from Sigma Aldrich. The plasmid pEGFP-N1 (Clontech Laboratories, USA), which contains the early promoter of HCMV and the enhanced green fluorescent protein (GFP) gene, was amplified in competent Escherichia coli and purified by using a NucleoBond® Xtra Midi (Clontech Laboratories, USA) kit. The purity of the DNA was confirmed by UV absorbance at 260/280 nm. DNA concentration was measured by UV absorbance at 260 nm.

Figure 1. Molecular structure of PEO113-b-PDEA50 (A), POEGMA70-b-P(OEGMA10co-DEA47-co-DPA47) (B) and BPEI (C).


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Solutions were prepared using ultrapure MilliQ® water. YOYO-1 was purchased from Life Technologies. All other reagents were purchased at the highest purity available.

2.2. Preparation of BPEI-free and BPEI-containing Polyplexes Calf thymus DNA was used in most of the measurements and pEGFP-N1 was employed in transfection experiments. Stock solutions were prepared in phosphate buffer saline (pH 7.4). DNA was labeled by using the intercalating dye YOYO-1 (dye molecules per DNA kbp = 50). The YOYO-1 labeled DNA or pEGFP-N1 solutions were added to determined amounts of block copolymer solutions in the same buffer to obtain polyplexes at N/P ratios 2, 5 and 10. The N/P ratio is expressed as the molar ratio of amine groups (from polymer) and phosphate groups (from DNA). The N/P ratio of the polyplexes was calculated based on the DEA unit (degree of protonation α = 0.44 at pH 7.4). The BPEI-containing polyplexes were prepared by using essentially the same procedure. In such a cases, the final step included the addition of desired amounts of BPEI to the previously prepared block copolymer polyplexes reaching final N/P ratios of 5 (for block copolymer polyplexes previously prepared at N/P = 2) and 10 (for block copolymer polyplexes previously prepared at N/P 5).

2.3. Physical Methods and Techniques Dynamic Light Scattering (DLS): DLS measurements were performed using an ALV/CGS-3 compact goniometer system consisting of a 22 mW HeNe linearly polarized laser operating at a wavelength of 633 nm, an ALV 7004 digital correlator and a pair of avalanche photodiodes operating in pseudo cross-correlation mode. The samples were placed in 10 mm diameter glass cells and maintained at a constant


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temperature of 25 ± 1 °C. The autocorrelation functions were based on 03 independent runs of 60 s counting time. The data were collected and further averaged by using the ALV Correlator Control software. The correlation functions were analysed using the nonlinear inverse Laplace transformation algorithm CONTIN resulting in distributions of relaxation times - A(τ ). The hydrodynamic radius (RH) of the nanoparticles was determined by using the straightforward Stokes-Einstein relation with D = τ -1q-2:

k BT q 2 RH = τ 6πη

(1)

kB is the Boltzmann constant, T is the absolute temperature, q is the scattering vector, η is the viscosity of the solvent and τ is the mean relaxation time related to the diffusion of the complexes.

Electrophoretic Light Scattering (ELS): ELS measurements were employed to determine the average zeta potential (ζ) of the supramolecular aggregates. The values were collected using a Zetasizer Nano-ZS ZEN3600 instrument (Malvern Instruments, UK). This instrument measures the electrophoretic mobility (UE) and converts the value to ζ-potential (mV) through Henry’s equation:

UE =

2 ε ζ f(ka) 3η

(2)

where ε is the dielectric constant of the medium and η is the viscosity. Furthermore, f(ka) is the Henry’s function, which was calculated through the Smoluchowski approximation f(ka) = 1.5. Each ζ-potential value reported in the manuscript is an average of 10 independent measurements with repeatability ± 2%.


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Isothermal Titration Calorimetry (ITC): The thermodynamic parameters of interaction between polyplexes and BPEI were determined through isothermal titration calorimetry (ITC) by using a MicroCal VP-ITC instrument. The reference cell was filled with water and the sample cell was filled with the block copolymer polyplexes at N/P = 5. The volume of both reference and sample cells was 1.4576 mL. The concentration of DNA at such condition was equal to 0.0525 mg.mL-1. The syringe was filled with 0.5 mg.mL1

of BPEI polymer solution. The titrations were performed by injecting 10 µL of BPEI

polymer solution in the sample cell every 600 s. The temperature was kept constant at 25 °C. The ITC raw data, which consists of peaks of power delivered to the sample cell during the titration, were integrated from a baseline to give the heat per injection as a function of N/P. The heat of dilution of the solution was determined as the heat of the last injections of the titration experiment and was subtracted from the interaction data. The set of data was fitted using the one-site model to extract the enthalpy of binding (∆H), binding constant (K) and the stoichiometry of binding (n) as previously described.26

2.4. Biological Assays Cell Culture: Telomerase immortalized rhesus fibroblasts (Telo-RF) (Princeton University, USA) were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% fetal bovine serum and antibiotic solutions (penicillin 10.000 µg.mL-1 and streptomycin 10.000 µg.mL-1) at 37ºC in CO2 atmosphere.


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Cell Viability: The cytotoxicity of the polyplexes and polymeric vectors was evaluated in Telo-RF cell line. The cells were seeded into 48-well plates at a density of 25000 cells/well growth for 24 h and afterwards treated with 200 µL of fresh DMEM plus 200 µL of solutions containing varying concentrations of polymeric vectors or polyplexes (produced with 1 µg of DNA). The cells were incubated for 24 h at 37 oC, washed with PBS and subjected to MTT assay. Briefly, 100 µL of MTT (3-(4,5-dimethylthiazol-2yl)2,5-diphenyl-tetrazolium bromide) reagent solution (0.5 mg.mL-1) was added to each well for 4 hs. The medium was aspirated and violet crystals of formazan generated by living cells at each well were dissolved in 100 µL of dimethyl sulfoxide (DMSO). The absorbance at 570 nm was measured using a Synergy microplate reader. The relative cell viability (%) was determined by comparing the absorbance at 570 nm with control wells containing untreated cells (100%).

Fluorescence Microscopy: Cellular uptake and gene expression were qualitatively examined by fluorescence microscopy. The Telo-RF cells were seeded on glass coverslips at a density of 50000 cells/well in the cell culture medium and incubated at 37ºC in CO2 atmosphere for 24 h. Next day, the cell culture medium was replaced by 200 µL fresh DMEM and 200 µL of solutions containing varying concentrations of polyplexes with 1 µg of YOYO-1 labeled DNA (cellular uptake experiments) or 1 µg of pEGFP-N1 (gene transfection experiments). After the incubation time (4 h for cellular uptake and 48 h for gene transfection experiments) the cells were washed three times with PBS buffer and stained with Hoechst Stain solution (20 µL Hoechst 1:1000) for 15 min to stain the cell nuclei. YOYO-1 labeled DNA or pEGFP-N1 are displayed in green and Hoescht stained nuclei appears blue. Images were acquired on a widefield Leica DMI 6000 B microscope (Leica Microsystems, Germany) coupled to an ultrafast Leica DFC365 FX digital camera (Leica Microsystems, Germany). In the acquisition of blue


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(Hoescht) and green (YOYO-1/GFP) the A4 (ex. 340-380, DC 400, em. 450-490) and L5 (ex. 460-500, DC 505, em. 512-542) cube filters were respectively selected.

Flow Cytometry: The cellular uptake and gene expression was further quantified by flow cytometry. The procedure was essentially the same employed to perform the imaging experiments except that after the incubation time the cells were washed with PBS buffer, trypsinized and resuspended in 150 µL PBS. The measurements were performed by using a BD FACS Canto II flow cytometer with further analysis using the Flowing software. The flow cytometry (10.000 events were collected for each experiment) acquisitions were performed in triplicate and the data are given as mean ± standard deviation.


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3. Results and Discussion 3.1. BPEI-free Polyplexes Recently, we demonstrated that the block copolymers PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) are able to properly condense DNA into nano-sized supramolecular aggregates with maximum dimensions that presumably enable their uptaken by cells via endocytic pathways. The calorimetric data revealed that the binding process is endothermic and therefore entropically driven due to counter ion release.26 The POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)/DNA binding takes place with higher thermodynamic outputs. Nevertheless, both nano polyplexes are highly stable in serum environment. Herein, we explore the biological assays of the potential non-viral vectors. The structural features (size and ζ-potential) of the produced polyplexes at different N/P ratios are summarized in Table 1 along to those produced by using BPEI (control system).

Table 1. RH and ζ-potential of the produced polyplexes at different N/P ratios.

N/P = 2

N/P = 5

N/P = 10

BPEI-free Polyplexes

PEO113-b-PDEA50

RH (nm)

ζ (mV)

RH (nm)

ζ (mV)

RH (nm)

ζ (mV)

78.5

+ 0.2

78.1

+ 1.7

78.9

+ 2.7

77.0

- 1.4

73.0

-0.6

72.5

+ 1.1

72.6*

+ 10.9

70.0*

+ 11.1

64.2*

+ 13.9

POEGMA70-b-P(OEGMA10co-DEA47-co-DPA47) BPEI

*measurements were acquired in water after adjusting the pH to 7.4.

The condensed structures have similar sizes (~ 64-80 nm) regardless the N/P ratio. The size slightly reduces as N/P ratio increases suggesting that the degree of


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compaction is enhanced in such direction. It is important to emphasize, however, that the

molecular

weight

of

POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)

is

substantially higher thereby suggesting a stronger binding and better compaction promoted by the polymer. The surface charge (ζ -potential) of the block copolymer polyplexes is essentially neutral becoming progressively more positive (or less negative) as the polymer amount increases. The nearly neutral surface charge is attributed to the steric shielding effect of PEO and PEOGMA. On the other hand, the ζ potential of the BPEI polyplexes is always positive reflecting the presence of protonated amine groups at the outer surface of the assemblies (~ 25% of nitrogen atoms of the BPEI polymer backbone are protonated at physiological pH).27,28 120

Control.

(A)

Polyplexes.

cell viability (%)

100

Polymeric Vector

80 60 40 20 0 Control.

(B)

Poliplexes.

100

Polymeric Vector

cell viability (%)

80 60 40 20 0 Control.

(C)

Polyplexes.

100

cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Polymeric Vector

80 60 40 20 0 l ro nt Co

P N/

2

P N/

2

P N/

5

P N/

5 P N/

10

P N/

10


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Figure 2. Telo-RF cell viability after 24 h incubation time with polymeric vectors or polyplexes: (A) POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47), (B) PEO113-bPDEA50 and (C) BPEI. The polyplexes were produced at different N/P ratios. The control refers to untreated cells. The results are expressed as mean ± SD (n = 3).

The cytotoxicity of the assemblies as well as of the free polymeric vectors was evaluated based on the MTT assay (whenever present, the amount of DNA - 1 µg - was kept fixed). The BPEI was used as a control system due to its well-known golden transfection efficiency, although linked to high levels of cytotoxicity. The results reported in Figure 2 evidenced that the block copolymer POEGMA70-b-P(OEGMA10co-DEA47-co-DPA47) is the less cytotoxic followed by PEO113-b-PDEA50 and ultimately BPEI. As expected, the cell viability progressively reduces as the polymer amount increases. For instance, at the highest evaluated N/P ratio (N/P = 10) the quantitative cell viability is respectively 95%, 70% and 58%. Additionally, the cell viability of the polyplexes is always higher than the monitored for the respective free polymeric vector (DNA-free system). This result is consistent with the residual positive charge at the polymer chains (partial protonation of amino groups). The positive charges promote cytotoxicity due to electrostatic interactions with a variety of negatively charged lipids present in several cell organelle21,29 thereby inducing, generally, cell apoptosis.30 Once complexation takes place, the positive charges are progressively neutralized by the phosphate groups of the nucleic acid, thus explaining the decreased cytotoxicity of the complexes. The cell viability profile suggests that amino groups (and particularly the degree of protonation) govern the cytotoxicity of the aggregates. Interestingly, the BPEI system has the lowest density of positive charges since only ~ 25% of nitrogen atoms at the


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polymer backbone are protonated at physiological condition (as stated before). Taking into account the same rationalization and the degree of protonation at physiological pH for PDEA (0.44) and PDPA (0.20) (See Figure S1), in the block copolymer systems roughly 50% of the nitrogen atoms are protonated. Accordingly, the cell viability seems not to depend on the overall positive charge concentration in the system (which is higher in the block copolymers at a given N/P ratio). It apparently reflects the charge distribution within the chains. Bearing in mind the molar ratio of protonated species per diamine group, the BPEI chains hold in average 1N+/172g.mol-1 whereas the values are ~ 1N+/650g.mol-1 and ~ 1N+/3000g.mol-1 respectively for PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47. Therefore, the cell viability is enhanced when charge distribution spreads over larger amounts of polymer. Presumably, the interaction of the catiomers and cell membrane perturbation effects might cause cell death. Nevertheless, the cytotoxicity of the polyplexes produced from PEO113-b-PDEA50

and

POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47

is

still

acceptable according to the ISO 10993-5:2009 since the cell viability was not reduced by more than 30% which would then be considered a cytotoxic effect. Subsequently, the cellular uptake behavior of the polyplexes produced at different N/P ratios was evaluated by means of flow cytometry and fluorescence microscopy (the DNA amount was kept fixed at 1 µg). The DNA was previously labeled by using the intercalating dye YOYO-1. The micrographs and representative raw flow cytometry data are respectively given in Figures 3 and 4.


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Figure 3. Fluorescence microscopy images of Telo-RF cells incubated with polyplexes at N/P 10 produced using YOYO-1 labeled DNA. The control refers to untreated Telo-RF cells.


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Figure 4. Flow cytometry data of untreated Telo-RF cells (A) and incubated with POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) (B), PEO113-b-PDEA50 (C) and BPEI (D) polyplexes at N/P 5 produced using 1 µg YOYO-1 labeled DNA.

It is well accepted that cellular uptake is markedly influenced by size, shape and surface charge of assemblies. The fluorescence microscopies convincingly demonstrate that the polyplexes are uptaken by the cells. In all cases, the green fluorescence coming from YOYO-1 labeled DNA is clearly observed, particularly in the cytoplasm. The quantitative data are revealed by the flow cytometry experiments with further data treatment. The 2D flow cytometry plot portrayed in Figure 4A reveals the autofluoresence of the cell line. The gate regions were selected based on the control sample and in such way that cellular debris of low forward scattering are avoided. The


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data treatment resulted in four quadrants where all the events located at the bottom-right quadrant were considered YOYO-1 positive healthy cells.31 The quantification of positive events (%) was based on the total number of events and they are given in Figure 5.

Untreated Cells1

Cell Uptake (% of positive events)

POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)

60

PEO130-b-PDEA50 BPEI

50 40 30 20 10

10

N

/P

5

2 /P

/P N

N

5

10

/P N

2 /P

/P N

N

5 /P

/P N

N

10

2 /P

N

on

tro

l

0

C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 5. Quantitative YOYO-1 positive Telo-RF cell percentage as given by the flow cytometry analysis after 4 hours incubation time with distinct polyplexes. The results are expressed as mean ± SD (n = 3).

The results evidence that cellular uptake is both polycation and N/P ratiodependent. The cellular uptake is enhanced as the N/P ratio increases. The highest percentage is achieved for cells treated with BPEI polyplexes where at N/P = 10 the percentage of YOYO-1 positive cells is over 60%. The behavior is certainly related to the presence of positive charges at the outer surface of the entities which enable favorable electrostatic interactions with cell membrane thereby smoothing the endocytic process. On the other hand, the percentage of positive events for PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)

polyplexes

is

smaller

when

compared with BPEI systems in the same N/P ratio (as also qualitatively noticed in Figure 3). The nearly electrostatically neutral assemblies disfavor the cellular uptake


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since the polyplex-membrane interactions take place via intermolecular forces of smaller magnitude (hydrophobic interactions, hydrogen bonding, etc.).21 The large exclusion volume promoted by the presence of the highly hydrated polymeric corona may also hamper the capturing. The values at N/P =10 are respectively 36% and 33% for PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47). This behavior initially suggest that polymer chains freely diffusing (but not taking part of the assemblies) may contribute to the process.18–20,31,32

The whole set of values,

nevertheless, are significantly different from those determined for the control. Despite the reduced cellular uptake of the block copolymer polyplexes, we highlight that the assemblies are less cytotoxic as compared to BPEI which is, indeed, one of the major barriers towards its applicability. However, in the step further, we have evidenced that the produced block copolymer polyplexes are very poor transfecting agents. These investigations were conducted by using the pEGFP-N1 plasmid instead of YOYO-1 labeled DNA. The pEGFP-N1 encodes the green fluorescent protein (GFP) which also allows gene expression quantification via flow cytometry analysis. The results are given in Figure 6. The percentage of GFP-positive cells never reached values higher than 3% and gene expression was not evidenced via microscopy analysis by using the block copolymers as transfecting agents. As a matter of comparison, the value determined for BPEI polyplexes at N/P 10 is 14%.


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Figure 6: Fluorescence microscopy images taking 48 hours post-transfection of TeloRF cells with POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47), PEO113-b-PDEA50 and BPEI polyplexes produced at N/P 10 using 1 µg of pEGFP-N1 along to respective flow cytometry quantifications.

Accordingly, the cellular uptake and gene expression results summarize that a reasonable amount of block copolymer polyplexes are endocytosed, however, gene release and/or expression do not take place efficiently. This suggests that the main barriers reside within the cells. Indeed, the endosomal escape is currently considered the major bottleneck of non-viral gene delivery.16 The polyplexes release is usually devoted to the “proton sponge” mechanism although this is currently a heavily debated hypothesis.17,33–35 Certainly, the presence of positive charges within the organelle lead to osmotic swelling, however, the herein reported results suggest that osmotic swelling


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may not be the key factor towards endosomal escape. It is well stablished that the endosome compartment is an acidic environment (pH < 5.5).36 Approximately ~ 50% of the nitrogen atoms of the BPEI polymer chains are protonated under such condition27,28 whereas the nitrogen atoms in the PDPA and PDEA units are fully protonated (See Figure S1). Hence, if one takes into account a fixed N/P ratio, the concentration of protonated nitrogen atoms is higher for polyplexes produced by using the amphiphilic block copolymers. Consequently, higher amount of water is expected to flow towards the vesicle in such systems aiding to its disruption. Conversely, the GFP-positive cells are substantially higher in cells treated with BPEI polyplexes. Indeed, Hennink et al. also demonstrated that other catiomers with osmotic swelling capacity have shown very low transfection efficiency.17,37 This highlights that intracellular trafficking and/or release mechanism of polyplexes still deserve discussions. In the next section we demonstrate that the cellular uptake and particularly the quantity of GFP-positive cells are substantially enhanced when the block copolymer polyplexes are produced and the systems are further supplemented by BPEI chains.

3.2. BPEI-containing Polyplexes Similarly to the performed characterization of the BPEI-free polyplexes, the size and surface charge of the mixed assemblies were determined as given in Table 2. The data indicate that the presence of BPEI induces slight increase in both size and surface charge of the manufactured aggregates.


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Table 2. Structural features (RH and ζ-potential) of the produced BPEI-containing polyplexes at different N/P ratios. N/P = 5

N/P = 10

PEI-containing Polyplexes

PEO113-b-PDEA50/BPEI

RH (nm)

ζ (mV)

RH (nm)

ζ (mV)

84.5

+ 2.1

86.1

+ 3.9

84.4

+ 0.57

87.7

+ 0.75

POEGMA70-b-P(OEGMA10-coDEA47-co-DPA47)/BPEI

This data suggest that when the block copolymer polyplexes are further supplemented by BPEI, the supramolecular aggregates are coated by the homopolycation. For instance, taking into account the block copolymer POEGMA70-bP(OEGMA10-co-DEA47-co-DPA47) and the final N/P 10, the presence of BPEI promotes an increase of about ~ 15 nm at the dimension of the assemblies (from 73.0 nm to 87.7 nm). Indeed, whenever BPEI is present, the dimension of the assemblies is larger regardless of the block copolymer or the N/P ratio. Similarly, the surface charge of the assemblies is slightly higher (about 2 mV) in the presence of BPEI. The strongest evidence on the interaction of BPEI with the previously produced polyplexes is demonstrated by the ITC data portrayed in Figure 7. In such experiments, the POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) and PEO113-b-PDEA50 polyplexes produced at N/P 5 were titrated using a stock BPEI solution up to the final N/P = 10 (NBPEI/P = 5). The integrated heat profiles qualitatively indicate that the interaction between the block copolymer polyplexes and BPEI is endothermic and therefore entropically-driven. This is an important remark indeed since the related literature reports that the BPEI/DNA interaction is exothermic.38,39 Accordingly, the ITC signals are presumably not related to BPEI-DNA interaction and the profiles are supposed to


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refer to the adsorption of BPEI chains onto the surface of the formerly produced block copolymer polyplexes. The integrated heat vs. NBPEI/P could be fitted using the one-site

kJ/mole of N

model and the obtained thermodynamic parameters are given in Table 3.

8000 7000 6000 5000 4000 3000 2000 1000 0

POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)/BPEI

PEO113-b-PDEA50/BPEI

1000 kJ/mole of N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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800 600 400 200 0 0

1

2

3

4

5

6

NBPEI/P Figure 7. ITC resulting integrated heat per mol of injectant (N) as a function of NBPEI/P along to fitting results (solid lines) for titration of POEGMA70-b-P(OEGMA10-coDEA47-co-DPA47) and PEO113-b-PDEA50 polyplexes with BPEI (block copolymer polyplexes were previously prepared at N/P = 5).


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Table 3. Thermodynamic and binding parameters obtained from ITC data. PEO113-b-

POEGMA70-b-P(OEGMA10-co-DEA47-

PDEA50/BPEI

co-DPA47)/BPEI

NBPEI

2.1

2.2

K (107 M-1)

17

8.6

∆H (kJ.mol-1)

971

7113

Parameter

The determined binding constants (K) evidence that BPEI binds stronger to PEO113-b-PDEA50 polyplexes whereas the enthalpy of binding is higher for POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47). The sources of endothermic heats are probably related to polymer conformational changes during the adsorbing processes. Additionally, repulsive interactions between the adsorbed surface molecules usually results in endothermic heats. Both processes, however, are entropically-driven and this is probably the result of the entropy increase of the water molecules upon desolvation of the chains. Nevertheless, the most important parameter regards to the stoichiometry of binding (N). The results portrayed in Table 3 evidence that the interaction of BPEI with the polyplexes takes place essentially up to NBPEI/P ~ 2 and further addition of the homopolycation does not evolve considerable heat variations meaning negligible interaction. This highlights that at final N/P 10 (NBPEI/P = 5) roughly 40% (molar percentage) of the added BPEI is coating the previously produced block copolymer polyplexes whereas about 60% (molar percentage) remains freely diffusing in the systems (uncomplexed fraction of chains). The biological assays of the mixed systems are portrayed in the sequence.


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120

Untreated Cells. POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)/BPEI

100

PEO113-b-PDEA50/BPEI BPEI

cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60 40 20 0 Control N/P 5

N/P 5

N/P 5 N/P 10 N/P 10 N/P 10

Figure 8. Telo-RF cell viability after 24 hours incubation time with BPEI-containing and pure BPEI polyplexes according to the legend. The control refers to untreated cells and the results are expressed as mean ± SD (n = 3).

The Figure 8 shows the cell viability after 24 hours of incubation with BPEIcontaining polyplexes. Not surprisingly, the cytotoxicity of the mixed systems is higher than the monitored for the pure block copolymer assemblies (Figure 2). However, the levels are smaller as compared to the cytotoxicity of BPEI polyplexes. Indeed, the main barrier for BPEI gene delivery is its high cytotoxicity level which hamper the clinical applications.6,37 It is evidenced that the % of viable cells incubated with mixed assemblies are always higher than 70% and this is not considered a cytotoxic effect according to the ISO 10993-5:2009. For instance, the cell viability of POEGMA70-bP(OEGMA10-co-DEA47-co-DPA47)/BPEI at N/P 10 is ~ 80% whist it is ~ 60% for pure BPEI at the same condition. Such pattern (mild cytotoxicity of the mixed polyplexes) is evidenced regardless the block copolymer or N/P ratio.


Biomacromolecules

Untreated Cells1 POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)

Cell Uptake (% of positive cells)

60

POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)/BPEI PEO130-b-PDEA50

50


40 30 20 10

/P 5 /P 10 N /P N 5 /P 10 N /P N 5 /P 10 N /P N 5 /P 10

N

N

10

/P

5

C

N

N

on t

/P

ro l

0

Figure 9. Quantitative YOYO-1 positive Telo-RF cell percentage as given by the flow cytometry analysis after 4 hours incubation time with distinct polyplexes according to the legend. The results are expressed as mean ± SD (n = 3).

The cellular uptake of the mixed assemblies was subsequently evaluated. The Figure 9 evidences that cellular uptake of the block copolymer polyplexes is enhanced in the presence of BPEI. The increase is steeper for POEGMA70-b-P(OEGMA10-coDEA47-co-DPA47), however, the pure BPEI polyplexes are still internalized to a higher extent as compared to any other produced system. Additionally, it should be emphasized again that the mixed systems are less toxic than the pure BPEI assemblies. The transfection efficiency data for the BPEI-containing polyplexes are reported in Fig. 10 (representative 2D cell distribution profiles are given in the Supporting Information File - Figure S2). 20 Untreated Cellsn

18

POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)/BPEI

16

PEO130-b-PDEA50

14


12 10 8 6 4 2

10 N/ P N 5 /P 10 N /P N/ 5 P 10 N/ P N 5 /P 10 N /P N 5 /P 10

/P

5 N

N

/P

ro l

0

Co nt

GFP Expression (% of positive cells)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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Figure 10. Quantitative GFP positive Telo-RF cell percentage as given by the flow cytometry analysis after 48 hours post-transfection with distinct polyplexes according to the legend (the polyplexes were produced using 1 µg pEGFP-N1 and the results are expressed as mean ± SD - n = 3).

Figure 10 demonstrates that gene expression is achieved when the block copolymer polyplexes are supplemented by BPEI chains. The values are still lower than those monitored for BPEI, however, we highlight once again that the cell viability levels of the mixed assemblies are higher. The levels of GFP expression reached by using the mixed assemblies are remarkable. These results essentially demonstrated that cytotoxicity and gene expression/gene delivery can be balanced by using a combination of less cytotoxic cargo agents (such as the investigated block copolymers) and a highly toxic counterpart (BPEI was currently used). These results are in agreement with previous researchers that have convincingly questioned the dominant role of the wellaccepted “proton sponge” effect in promoting gene transfer, thereby proposing the central importance of uncomplexed polycation chains as an alternative explanation.15– 18,29,38

These claims are also supported by the gene transfection data of polyplexes

produced at NBPEI/P = 2 (final N/P = 7) as given in the Supporting Information File (Figure S3). At this condition, according to the ITC data (Figure 7 and Table 3), the added PEI all adsorbed onto the surface of the previously produced block copolymer polyplexes therefore making part of the assemblies. The gene expression at N/P =10 (in the presence of uncomplexed chains) is substantially higher than at N/P = 7 (in the presence of only adsorbed BPEI chains) thus suggesting that, indeed, the major contribution for enhanced gene transfection is the presence of freely diffusing (uncomplexed) chains.


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Overall, these results suggest that it may not be necessary to invest much effort in novel cationic polymers for gene delivery since one of the best transfecting agents is already known (BPEI). The issue, however, regards to its degree of cytotoxicity. Therefore, the combination with less cytotoxic cargo agents may be a clever strategy. The cellular uptake, subcellular localization and gene expression of the mixed assemblies were also examined by fluorescence microscopy (Figures 11 and 12).

Figure 11. Fluorescence microscopy images of Telo-RF cells incubated with mixed polyplexes at N/P 10 produced using YOYO-1 labeled DNA. The control refers to untreated Telo-RF cells.


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Figure 12: Fluorescence microscopy images taking 48 h post-transfection of Telo-RF cells with POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)/BPEI and PEO113-bPDEA50/BPEI polyplexes produced at N/P 10 using 1 µg of pEGFP-N1.

Although the microscopies does not allow for quantitative analysis, the Figure 11 demonstrates at least qualitatively that the mixed assemblies are uptaken to a higher extent as compared to the pure block copolymer polyplexes (Figure 3). In Figure 11, the YOYO-1 labeled DNA is spread throughout the cells and the green fluorescence is also clearly observed inside de nucleus as represented by the dot-formed green spots (the arrows indicate some cells with dot-formed green spots representing labeled DNA inside de nucleus). The results convincingly evidence that DNA was translocated to the


Biomacromolecules

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nucleus. As a matter of comparison, dot-formed green spots inside the nuclei are barely seen for the pure block copolymer polyplexes (Figure 3). Such evidence implies good predisposition for effective transfection which was indeed also qualitatively confirmed by the images taking post-transfection using pEGFP-N1 (Figure 12). Finally, we underline that similar investigations were also conducted by using HeLa cells. The importance of uncomplexed polymer chains in cellular uptake and particularly in gene expression was evidenced whatever the cell line. This complementary data are given in the Supporting Information File (Figures S4-S6).

Conclusions These investigations highlight that the block copolymers PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) are able to condense DNA into nanosized polyplexes. The complexes are marginally cytotoxic and they are uptaken by the cells to some extent. On the other hand, the GFP expression assays demonstrated that DNA delivery is negligible, meaning that intracellular trafficking hinders efficient release of the genetic material. It was found that the cellular uptake, and particularly the quantity of GFP-positive cells, is substantially enhanced when the block copolymer polyplexes are produced and further supplemented by BPEI chains. The scattering measurements confirmed that BPEI chains adsorb onto the surface of the preformed polyplexes leading to increase in the overall size and surface charge, although most of the added BPEI chains remain freely diffusing (uncomplexed) as suggested by the ITC data. Such evidences imply that enhanced cellular uptake is smoothed by the adsorption of BPEI chains. The mixed assemblies are slightly more cytotoxic than the pure counterparts. Principally, the presence of free (or weakly bound) highly charged polymer chains plays a central role in gene transfection and intracellular trafficking.


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Taking into account the highly charged BPEI chains present in the systems, the osmotic swelling is certainly enhanced inside the organelles, however, it may not induce the gene release and the escape is supposed to be essentially governed by the ability of the uncomplexed chains to disrupt the endosomal membrane thereby preventing the entrapment of the entities into lysosomes. Accordingly, the transfection efficiency vs. cytotoxicity issue might, at least to some extent, be balanced by a mixture of BPEI and less cytotoxic agents such as for instance by using the proposed block copolymers.

Associated Content The Supporting Information is available free of charge on the ACS Publications website: Molar ratio of deprotonated species per diamine group (NR3) as a function of pH for PDPA and PDEA, complementary gene expression data of mixed systems at N/P 7, biological assays in HeLa cells. Author Information Corresponding Author * Universidade Federal do ABC, Av. dos Estados 5001, Santo André - SP, Brazil. E-mail: [email protected] ORCID Fernando Carlos Giacomelli: 0000-0002-6872-9354 Notes The authors declare no competing financial interest.

Acknowledgements This work was sponsored by FAPESP (Grant No. 2014/22983-9). F.C.G acknowledges the productivity research fellowship granted by CNPq (Grant No.


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302467/2014-9) and C.E.C thanks the fellowship granted by FAPESP (Grant No. 2015/24686-4). The CEM at UFABC is acknowledged for the accessibility to the Malvern light scattering equipment. P.I.R, V.S. and C.G. are grateful to FAPERGS, CNPq and UFSM.

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TOC - Graphical Abstract

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Gene Transfection Mediated by Catiomers ... - ACS Publications

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