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Ef#cient Condensation of DNA into Environment-Responsive Polyplexes Produced from Block Catiomers Carrying Amine or Diamine Groups Lindomar Jose Calumby Albuquerque, Kelly Annes , Marcella Milazzotto, Bruno Mattei, Karin A. Riske, Eliézer Jäger, Jiri Panek, Petr Stepanek, Peter Kapusta, Paulo Muraro, Augusto de Freitas, Vanessa Schmidt, Cristiano Giacomelli, Jean-Jacques Bonvent, and Fernando Carlos Giacomelli Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04080 • Publication Date (Web): 18 Dec 2015 Downloaded from http://pubs.acs.org on December 22, 2015

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Efﬁcient Condensation of DNA into Environment-Responsive Polyplexes Produced from Block Catiomers Carrying Amine or Diamine Groups

Lindomar J. C. Albuquerque,† Kelly Annes,†Marcella P. Milazzotto,†Bruno Mattei,‡ Karin A. Riske,‡ Eliézer Jäger,§ Jiří Pánek, § Petr Štěpánek, § Peter Kapusta, ╨ Paulo I. R. Muraro,# Augusto G. O. De Freitas,# Vanessa Schmidt, # Cristiano Giacomelli,# JeanJacques Bonvent,† 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. §

Institute of Macromolecular Chemistry AS CR, Prague, Czech Republic.

╨ #

J. Heyrovsky Institute of Physical Chemistry, Prague, Czech Republic.

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 intracellular delivery of nucleic acids requires a vector system as they cannot diffuse across lipid membranes. Although polymeric transfecting agents have been extensively investigated, none of the proposed gene delivery vehicles fulfills all the requirements needed for an effective therapy namely ability to bind and compact DNA into polyplexes, stability in serum environment, endosome-disrupting capacity, efficient intracellular DNA release and low toxicity. The challenges are mainly attributed to the conflicting properties such as for instance stability vs. efficient DNA release and toxicity vs. efficient endosome-disrupting capacity. Accordingly, investigations aiming at safe and efficient therapies are still essential to achieve clinical success of gene therapies. Taking into account the mentioned issues, herein it has been evaluated the DNA condensation ability of

poly(ethylene oxide)113-b-poly[2-

(diisopropylamino)ethyl methacrylate]50 (PEO113-b-PDPA50), poly(ethylene oxide)113-bpoly[2-(diethylamino)ethyl methacrylate]50 (PEO113-b-PDEA50), poly[oligo(ethylene glycol)methyl

ether

methacrylate]70-b-poly[oligo(ethylene

methacrylate10-co-2-(diethylamino)ethyl methacrylate47] poly[oligo(ethylene glycol)methyl

glycol)methyl

methacrylate47-co-2-(diisopropylamino)ethyl

(POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) glycol)methyl ether

ether

ether

methacrylate]70-b-poly{oligo(ethylene

methacrylate10-co-2-methylacrylic

(dimethylamino)ethyl)methylamino]ethyl

and

ester44}

acid

2-[(2-

(POEGMA70-b-P(OEGMA10-co-

DAMA44). The block copolymers PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10co-DEA47-co-DPA47) were evidenced to properly condense DNA into particles with desirable size for cellular uptake via endocytic pathways (RH ~ 65-85 nm). The structure of the polyplexes was characterized in details by scattering techniques and atomic force microscopy. The isothermal titration calorimetric data revealed that the polymer/DNA


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binding is endothermic and therefore the process in entropically driven. The combo of results supports that POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) condenses DNA more efficiently and with higher thermodynamic outputs as compared to PEO113-bPDEA50. Finally, circular dichroism spectroscopy indicated that the conformation of DNA remained the same after complexation and that the polyplexes are highly stable in serum environment.


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1. Introduction The quick advances in nanotechnology and their applications in medicine recently opened a new and promising area of scientific investigations called nanomedicine. In this field, materials at the nanoscale are employed to deliver active molecules, genes and imaging agents into target sites. The increasing interest in nanomedicine is driven essentially by the emerging successes of nanoparticle based delivery systems, which have manifested a number of benefits compared to the administration of the free active agents. The advantages include protection of encapsulated substances against degradation, higher therapeutic efficacy, longer blood circulation half-life and ideally, targeting without damaging healthy tissues.1–3 Nevertheless, low molecular weight active agents can be generally delivered in the extracellular milieu of damaged tissue to further freely diffuse through the plasma membrane. On the other hand, the delivery of genes (nucleic acids) requires the cellular internalization which is generally difficult due to their ionic characteristic.4 Cellular internalization of naked DNA lacks efficiency due to its negative charge since the anionic phosphate backbone is electrostatically repelled by the negatively charged plasma membrane. Furthermore, whenever DNA is directly administered into the bloodstream, it is rapidly eliminated mainly by DNase attack.5,6 Therefore, the development of DNA cargo delivery systems is expected to provide protection and stability to such a biomacromolecule allowing the cell internalization. Accordingly, the success of gene therapy largely depends on the development of efficient delivery vectors. Currently, investigations are focused mainly into two classes: viral and nonviral cargos. The viral vectors, such as retroviruses and adenoviruses, are efficient gene delivery systems because of their high transfection capability. On the other hand, there are serious drawbacks associated with them including limited DNA


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carrying capacity, difficulties in scaling-up production, possible introduction of immunogenic and inflammatory response as well as gene mutation.7–10 The concerns related to the safety and immunogenicity of viral vector have created a need for the investigation of synthetic vectors. In this regard, cationic polymers that can electrostatically bind to phosphodiester backbones of nucleic acids to form polyion complexes (polyplexes) represent promising class of DNA delivery systems.11 Amongst synthetic vectors, the polycation polyethylenimine (PEI) has been shown to electrostatically interact with DNA at the nanoscale and the final polyplexes can be internalized by cells with high efficiency.12,13 Furthermore, PEI in the branched configuration exhibits a pH buffering effect which allows endosomal disruption after endocytosis since the acidic late endosome (pH ~ 5) allows a higher degree of PEI protonation which according to the proton sponge hypothesis leads to the swelling and disruption of the organelles consequently releasing the DNA into the cytoplasm.14 This prevents DNA from accumulation and enzymatic digestion in lysosomes and might explain the high transfection efficiency of PEI-based polyplexes. Nevertheless, the excess of cationic charges in PEI chains frequently leads to high levels of toxicity, which is one of the major limiting factors for its use in vivo.15 Besides, the positively charged PEI-based polyplexes are generally attracted by negatively charged serum proteins leading to short blood circulation time.16,17 The abovementioned drawbacks can be reduced by providing a steric shield to the polyplexes by the conjugation of poly(ethylene glycol) (PEO) to the polycation.18,19 The stabilization of polyplexes by a PEO shell reduces their toxicity and enables the nanostructures to circulate in the bloodstream for longer periods.20 However, these strategies generally reduce transfection efficiency.19,21 An alternative strategy to overcome such disadvantage is the usage of polycations with more broadly spread


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charge distribution along the polymer chain since the toxicity is directly related to charge density. In this regard, poly(2-(dimethyl amino)ethyl methacrylate) (PDMA) and poly(2-(diethyl amino)ethyl methacrylate) (PDEA) can be of interest as the charge density is lower than in PEI, thus imparting lower cytotoxicity to the former. PDMA and PDEA are nevertheless sufficiently protonated at physiological pH (pKa values 7.4 and 7.5 respectively)22 to enable DNA complexation and the higher degree of protonation at lower pH allows endosomal escape. However, the DNA release was demonstrated to slow down after cell internalization.23 Consequently, the production of cationic polymers carrying diamine groups with different pKa values have been considered to increase the ability to destabilize the endosome and positively affect the transfection levels.24,25 Therefore, taking into account that balance and optimization between toxicity and transfection efficiency is still an issue to be overcome, the goal of present study was to move forward in the topic by investigating the DNA binding ability, the thermodynamics and the structure of polyplexes produced from PEO- and POEGMAbased block copolymers linked to different polyamine chains. The complexation of calf thymus DNA (ctDNA) with the block copolymers PEO113-b-PDPA50, PEO113-bPDEA50, POEGMA70-b-P(OEGMA10-co-DAMA44) and POEGMA70-b-P(OEGMA10co-DEA47-co-DPA47) was investigated. The block copolymers POEGMA70-bP(OEGMA10-co-DAMA44) and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) hold diamine groups (Figure 1). The one with higher pKa is supposed to participate in the ion complex formation with ctDNA whereas the one with lower pKa is expected to participate of the enhanced intracellular activity of DNA through the buffering capacity in the endosomal compartment. The formation of polyplexes has been probed by light scattering (SDELS), small-angle X-ray scattering (SAXS) as well as atomic force


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microscopy (AFM). ctDNA condensation whenever evidenced has been suggested by agarose gel electrophoresis and fluorescence lifetime correlation spectroscopy (FLCS). Possible conformational changes on the ctDNA chains due to complexation were monitored via circular dichroism (CD) and UV-vis spectroscopy.

2. Experimental 2.1. Chemicals and Samples: Block copolymer samples used in this study were synthesized by either atom transfer radical (ATRP) or reversible addition-fragmentation chain transfer (RAFT) polymerization techniques using already well-established protocols. Hereinafter, the subscripts refer to the mean degree of polymerization of each monomer in the copolymers. Poly(ethylene oxide)113-b-poly[2-(diisopropylamino)ethyl methacrylate]50 (PEO113-b-PDPA50, Mn = 15700 g.mol-1, Mw/Mn = 1.20) and poly(ethylene

oxide)113-b-poly[2-(diethylamino)ethyl

methacrylate]50

(PEO113-b-

PDEA50, Mn = 14500 g.mol-1, Mw/Mn = 1.20) were synthesized following the procedures described by Liu et al.26 with adaptations as described in details elsewhere.27 Poly[oligo(ethylene glycol)methyl

glycol)methyl

ether

ether

methacrylate]70-b-poly[oligo(ethylene

methacrylate10-co-2-(diethylamino)ethyl

(diisopropylamino)ethyl methacrylate47]

methacrylate47-co-2-

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

DPA47), Mn = 59000 g.mol-1, Mw/Mn = 1.28) and poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly{oligo(ethylene glycol)methyl ether methacrylate10-co-2methylacrylic

acid

2-[(2-(dimethylamino)ethyl)methylamino]ethyl

ester44}

(POEGMA70-b-P(OEGMA10-co-DAMA44), Mn = 49100 g.mol-1, Mw/Mn = 1.38) were synthesized by RAFT polymerization in toluene employing a one-pot/two-step approach involving

the

sequential

addition

of

monomers

and

4-cyano-4-

[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid chain transfer agent, as adapted


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from a recent study by our group.28 DAMA monomer was synthesized following the exact same procedure reported by Funhoff et al.29 The molecular structure of copolymer samples used in the investigations is shown in Figure 1. Details of synthesis procedures as well as the NMR spectra and GPC chromatograms of all the polymers are provided in the Supporting Information File (Figures S1-S5).

(A)

(B)

(C)

(D)

Figure 1. Molecular structure of the block copolymers: PEO113-b-PDPA50 (A), PEO113b-PDEA50 (B), POEGMA70-b-P(OEGMA10-co-DAMA44) (C) and POEGMA70-bP(OEGMA10-co-DEA47-co-DPA47) (D).

Solutions were prepared using ultrapure MilliQ® water. YOYO-1 was purchased from Life Technologies. All the other chemicals were purchased at the highest purity available from Sigma-Aldrich and used as received.

2.2. Preparation of Polymer/DNA Polyplexes: The polyplexes have been prepared by firstly dissolving DNA from calf thymus (ctDNA) in phosphate buffer saline (pH 7.4). The stock ctDNA solution was added to determined amounts of polymer solutions in the


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same buffer to obtain particles with different N/P ratios which is expressed as the ratio of moles of the amine groups (from polymer) to phosphate groups (from the DNA). The N/P ratio for POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) was calculated based only on the DEA unit whereas for POEGMA70-b-P(OEGMA10-co-DAMA44) it was consider one polymer nitrogen atom per DAMA unit since the second amine atom is essentially deprotonated at pH 7.4 (degree of protonation α = 0.012).

2.3.

Characterization

The DNA condensation has been investigated by agarose gel electrophoresis, fluorescence lifetime correlation spectroscopy (FLCS) and isothermal titration calorimetry (ITC). The structural characterization of the produced polyplexes was evaluated by a variety of techniques namely light scattering (dynamic, static and electrophoretic light Scattering), small angle X-ray scattering (SAXS) and atomic force microscopy (AFM). Circular dichroism spectroscopy (CD) has been finally employed to probe possible conformational changes due to DNA condensation. The technical details regarding equipments, data acquisition and data treatment are given in the Supporting Information File.

3.

Results and Discussion

3.1.

DNA Binding and Condensation The binding capability of cationic polymers to DNA is a prerequisite in gene

delivery systems.

Herein, the binding capacity of the block copolymers has been

investigated qualitatively via agarose gel electrophoresis and quantitatively by fluorescence lifetime correlation spectroscopy (FLCS). The gel retardation assays for the whole set of block copolymers in increasing N/P ratios is portrayed in Figure 2.


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1

2 3

4

5

6

7

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8

9

10

(A)

1

2

3

4

5

6

7

8

9

10

(B)

1

2

3

4

5

6

7

8

9

10

(C)

1

2

3

4

5

6

7

8

9

10

(D)

Figure 2. Agarose gel electrophoresis data for PEO113-b-PDPA50 (A), POEGMA70-bP(OEGMA10-co-DAMA44) (B), PEO113-b-PDEA50 (C) and POEGMA70-b-P(OEGMA10co-DEA47-co-DPA47) (D) at different N/P ratios: ladder (1), ctDNA (2), N/P = 0.25 (3), N/P = 0.50 (4), N/P = 0.75 (5), N/P = 1.00 (6), N/P = 2.00 (7), N/P = 3.00 (8), N/P = 4.00 (9), N/P = 5.00 (10).


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The lane 1 is the illumination image of ctDNA ladder marker where lane 2 is the image of ctDNA intercalating with GelRedTM (N/P = 0). This is a negative control where only ctDNA is present. Regarding PEO113-b-PDEA50 (C) and POEGMA70-bP(OEGMA10-co-DEA47-co-DPA47) (D) the illuminating intensity decreased from lanes 3 to 10 with increasing N/P ratio. Indeed, at N/P = 0.50 and beyond, there is a blocking of illuminating intensity and it thus evidences that the block copolymers were able to bind to and completely neutralize the negative charges of ctDNA. The polyplexes appeared to be essentially shielded from the electric potential and remained static under electric field. On the other hand, the experimental data also evidence that POEGMA70b-P(OEGMA10-co-DAMA44) and PEO113-b-PDPA50 are poor complexers where the ctDNA negative charges seems not to be efficiently neutralized and therefore illuminating intensity remains even at high N/P ratios. Subsequently,

fluorescence

lifetime

correlation

spectroscopy

(FLCS)

experiments were performed to quantify the ctDNA condensation. For these experiments, ctDNA was labeled with YOYO-1, a DNA-intercalating fluorophore. YOYO-1 is virtually non-fluorescent in aqueous buffer and becomes highly fluorescent upon binding to DNA. FLCS then monitors exclusively the fluorescence intensity fluctuations caused by ctDNA molecules randomly diffusing through (in our case: also undergoing conformational changes) in the confocal detection volume. The FLCS autocorrelation function (ACF) for the simplest case of one diffusing component is mathematically given by Equation 1.

G (t ) =

1 Np

  1   2 1/ 2   (1 + t / τ D )(1 + t / k τ D ) 


(1)

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(A)

N/P = 0.0 N/P = 0.3 N/P = 0.8 N/P = 1.0 N/P = 1.6 N/P = 2.1 N/P = 2.6 N/P = 3.4 N/P = 3.8

0.3

G (t)

0.2

0.1

0.0 -3

10 8

10

-2

-1

10

0

10

1

10

2

10

2

10

2

10

2

10

10

3

10

4

3

10

3

10

3

10

correlation time (ms) (B)

G (t)

6

4

2

0 -3

10

-2

10 10 10 10 correlation time (ms)

-3

10

-2

10

10

-1

0

1

4

(C) 18 15

G (t)

12 9 6 3 0 10

-1

0

10

1

10

10

4

correlation time (ms) (D) 18 15 12 G (t)

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

9 6 3 0 10

-3

10

-2

-1

10

10

0

1

10

10

4

correlation time (ms)

Figure 3. Autocorrelation functions for ctDNA 1 nM intercalated with YOYO-1 (cdye/kbp = 50) complexed with different block copolymers as a function of N/P ratio: PEO113-bPDPA50 (A), POEGMA70-b-P(OEGMA10-co-DAMA44) (B), PEO113-b-PDEA50 (C) and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) (D). (kbp means kilo base pairs)


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wherein Np is the average number of diffusing fluorescent particles in the confocal volume, t is the correlation time, τD refers to the residence time in focus and k is the ratio of axial to radial radii of the confocal volume. In this relationship, the average number of fluorescent particles (Np) in the detection volume is inversely proportional to the y-intercept (t=0) of the autocorrelation function. Np is a very robust experimentally observable parameter. Its determination does not need any special mathematical treatment of the ACF, like e.g. diffusion model fitting and this means it is independent on the details of the diffusion kinetics. Before condensation, multiple YOYO-1 molecules intercalated into the extended flexible ctDNA chain behave like single, individual emitters, although their diffusion is obviously restricted. Upon condensation, single YOYO-1 molecules inside one ctDNA molecule are not distinguishable from each other. A compacted domain, or, in an extreme case, a fully condensed ctDNA molecule behaves like a single bright particle.30 Thus, a reduction in Np, the average observable number of fluorescent particles is expected as the condensation proceeds. In terms of experimental observables, this means increase of the correlation amplitude of the obtained ACFs as polyamines are added. The FLCS raw data are depicted in Figure 3. The fluorescence correlation curves were monitored as a function of increasing polyamine amount (N/P ratio). Strong evidence of ctDNA condensation is a pronounced increase in the FLCS correlation amplitude observed for PEO113-b-PDEA50 (C) and POEGMA70-b-P(OEGMA10-coDEA47-co-DPA47) (D). Figure 4 portrays the determined average number of particles (Np) as a function of N/P during the complexation by using different polyamine polymers according to the legend. It is worth mentioning that ctDNA molecules are not uniform, because they differ in length of nucleotide sequence. Consequently, the labeling by YOYO-1 is not uniform, either. The initial Np value (at N/P = 0) differs


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from system to system. Accordingly, this starting Np has been normalized to 1.0 in Figure 4 in order to enable the comparison between all investigated complexing agents. The results are thus discussed as relative condensation. 1.0 Normalized Particle Number

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0.8

0.6

0.4

0.2

0.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 N/P

Figure 4. Normalized Particle Number (Np) as a function of N/P during ctDNAcomplexation using different polyamine polymers: PEO113-b-PDPA50 (■), PEO113-bPDEA50 (●), POEGMA70-b-P(OEGMA10-co-DAMA44) (▲) and POEGMA70-bP(OEGMA10-co-DEA47-co-DPA47) (○).

The magnitude of Np reduction as a function of N/P is a direct measure of the efficiency of a DNA-condensing agent. Figure 4 convincingly reports that, e.g., PDPA is a poor DNA-complexing agent. The pKa of the diisopropylamino group of the PDPA block was determined to be equal to 6.8.31 This is defined as the pH at which 50% of the amino groups in the polymer chains are protonated. The degree of protonation (α) of amino groups can be determined using the following relation:

α=

([H 3 0 + ] / K a ) (1 + [H 3 0 + ] / K a )

(2)

accordingly, the degree of protonation of PDPA at pH = 7.4 is about 0.19 and then the inability to properly condense the DNA chains may be attributed to insufficient degree of ionization at the physiological condition. The condition therefore does not provide


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strong electrostatic repulsion between PDPA segments within the polymer chains and thus its reasonable collapsed conformation does not favor electrostatic attraction with the ctDNA chains of the surroundings. On the other hand, at pH 7.4 using a pKa of 7.5 we estimated α = 0.56 for PDEA. Consequently, a more extended conformation is expected in PDEA-containing polymers enabling electrostatic association of ctDNA chains. Comparing the experimental data, one can notice that the more efficient complexation occurs by using POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47). It happens faster compared to PEO113-b-PDEA50 and the condensation seems to be completed at N/P ~ 0.5. The result may be attributed to an additional small contribution of the protonated amino groups from the PDPA (α = 0.19) in the condensation process. The experimental data related to POEGMA70-b-P(OEGMA10-co-DAMA44) surprised us as it evidenced that the block copolymer is incapable to efficiently condense ctDNA. The result is puzzling since the PDAMA block has two amino groups with pKa1 = 9.3 and pKa2 = 5.5. The amino group with higher pKa is expected to give the polymer the capability to properly condense ctDNA at physiological pH once the complexation of the homopolymer PDAMA with DNA was demonstrated.29 This experimental data indicates that due to reasons that have to be further investigated, the additional polymer segment in the complexing unit deeply interferes in the binding affinity of PDAMA as the complexation with ctDNA was hindered by the presence of POEGMA. It is definitely our interest to further deeply understand the reasons behind such behavior. To this end, it is worth highlighting that the Np parameter, which is a reliable readout FLCS parameter, accurately shows differences in the promoted degree of complexation. The technique provides detailed information on DNA interaction with gene carriers which is crucial to the development of safe and effective nonviral gene delivery vectors. Additionally, the raw FLCS data showed practically invariant count


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rate during the condensation process indicating that dye release does not interfere in the phenomenon. Furthermore, the brightness of the fluorescent molecules increases in the presence of continuously higher amounts of PEO113-b-PDEA50 and POEGMA70-bP(OEGMA10-co-DEA47-co-DPA47) which is indeed a complementary indication of DNA complexation (data not shown). In view of that, since gene carriers requires long circulation at the bloodstream at a slightly basic condition, and taking into account that ctDNA chains are not efficiently condensed by PEO113-b-PDPA50 and POEGMA70-b-P(OEGMA10-co-DAMA44) as convincingly demonstrated by either agarose gel electrophoresis or FLCS, the further characterization were focused on PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10co-DEA47-co-DPA47) polyplexes.

3.2.

Structural Evolution during Polyplexes Formation Probed by DLS and ELS The DNA complexation was also scanned as a function of the N/P ratio by

electrophoretic and dynamic light scattering. The intensity and volume distributions of size for pure calf thymus (ctDNA) at 0.10 mg.mL-1 is given in Figure S6A (Supporting Information File). The intensity distribution is bimodal with populations centered at 16.9 nm and 392.0 nm. Although the presence of aggregates is shown in the intensity distribution of sizes, its relative amount is substantially small since the presence of aggregates is no longer seen in the volume distribution. This is also suggested by the corresponding AFM image (Figure S6B). Therefore, one can consider that the majority of ctDNA in solution behaves as freely diffusing chains. Taking into account that ctDNA is highly polydisperse in nature, one should not be surprised with such experimental evidence. Regarding PEO113-b-PDPA50 and POEGMA70-b-P(OEGMA10co-DAMA44), the condensation is not efficient as already evidenced. The distribution of sizes at polymer/ctDNA ratio N/P = 5.0 is representatively portrayed for POEGMA70-b-


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P(OEGMA10-co-DAMA44) in Figure S7. The experimental data clearly evidences that the copolymer is a poor complexer as the distributions of size related to the presence of free ctDNA and aggregates are still observed even at such high N/P ratio (N/P = 5.0). Analogous behavior was experimentally observed for PEO113-b-PDPA50 (not shown here). Similarly, the Figure S8 shows the distribution of sizes for PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) complexed with ctDNA at different N/P ratios. Considering PEO113-b-PDEA50, it can be seen that at N/P ≥ 1.0 only a single population of particles is present whereas for N/P < 1.0, although it progressively became less visible, the size distribution of free ctDNA can still be seen along to the distribution at ~ 100 nm related to the formation of the polyplexes. This means that free unbound ctDNA chains still coexist with the supramolecular aggregates. Yet again, the behavior was similarly observed during the complexation with POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47). In that case, however, already at N/P = 0.5 the distribution related to free ctDNA chains is no longer seen (Figure S8B). The Figure S8 also reveals that the distribution related to the formation of polyplexes shifts progressively towards the left-hand side which accordingly means a compaction of the structure as the polymers are continuously added. This is quantitatively portrayed in Figure 5 which reports the values of size (RH) and ζ-potential monitored during the complexation as a function of N/P for both complexers. The sizes indeed reduce from N/P = 0.0 to N/P ~ 2.0 and afterwards it reaches a nearly constant value. The polyplexes produced from POEGMA70-b-P(OEGMA10-co-DEA47-coDPA47) are smaller than those produced from PEO113-b-PDEA50 regardless the N/P ratio up to N/P = 2.0. Beyond that, they roughly hold the same size (RH ~ 70 nm) although


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the

molecular

weight

of

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

is

substantially higher as compared to PEO113-b-PDEA50.

(A) 160

RH (nm)

140

120

100

80

60 0

1

2

3

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5 0 ζ-potential (mV)

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-5 -10 -15 -20 -25 -30 0

1

2

3

4

5

N/P

Figure 5. RH (A) and ζ-potential (B) of the polyplexes as a function of N/P ratio: PEO113-b-PDEA50 (●) and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) (○).

The values of ζ-potential are reported in Figure 5B. The ζ-potential of free ctDNA is negative and related to the presence of naked phosphate groups. The addition of the copolymers to ctDNA induced the condensation of the biomacromolecule, which resulted in an increase in the ζ-potential. The values become less negative with increasing N/P ratio reaching an asymptotic value at ζ ~ 0 mV for N/P ≥ 2.0 with no further significant variations. The increase is more rapidly reported for POEGMA70-bP(OEGMA10-co-DEA47-co-DPA47) compared to PEO113-b-PDEA50 although the final values are essentially the same. At higher N/P ratios the ζ-potential still remains close to


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0 mV which is attributed to the presence of the stabilizing PEO or POEGMA shells at the outer surface of the produced aggregates. Although the whole set of data so far reported indicates that at N/P = 1.0 the ctDNA chains are completely condensed by the block copolymers, in general, higher N/P ratios are necessary to form stable polyplexes that are appropriate for gene transfection. The results reported hereafter demonstrates that at N/P = 2.0 both block copolymers are able to condense ctDNA and furthermore the supramolecular assemblies are highly stable in serum environment. Additionally, taking into account that the amount of polymer in the assemblies should ideally be the lowest possible to avoid cytotoxicity related to the presence of excise amounts of positively charged entities, it has been performed a detailed structural characterization of the entities at N/P = 2.0.

3.3

Detailed Scattering and Imaging Characterization at N/P = 2.0 The detailed characterization of the produced supramolecular aggregates at N/P

= 2.0 have been performed by using the combination of static and dynamic light scattering along to small-angle x-ray scattering (SAXS) and imaging (AFM). The Figure 6 portrays the static light scattering (1/Isc (q) vs. q2)) (A and D) and autocorrelation functions along to the respective distributions of relaxation times (B and E)

for

PEO113-b-PDEA50

and

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

polyplexes. The values of radius of gyration (RG) were estimated from the slopes of the SLS curves and the DLS data provided informations on the hydrodynamic radius and size dispersity. The supramolecular aggregates are characterized by a single particle population. The cumulant expansion fitted the curves reasonably well suggesting monomodal distributions of size (µ2/Γ 2 < 0.15). This is also supported by the distribution of relaxations times of the autocorrelation functions. The single relaxation


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mode (Γ= 1/τ ) was plotted as a function of q2 (Figure S9). The linear Γ vs. q2 behavior was evidenced to be reproducible and it is therefore representative of a diffusive mode. The mean hydrodynamic radius (RH) of the polymer/ctDNA polyplexes could then be determined from the slopes of the Γ vs. q2 plots by further using the straightforward Stokes-Einstein relation. The average radii (RH) were determined as being respectively equal to 72.9 nm and 77.0 nm for PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10co-DEA47-co-DPA47) whereas RG determined from static light scattering are equal to 75.3 nm and 85.0 nm. Taking into account the values of RG and RH the structuresensitive parameters (ρ = RG/RH) were determined (RG/RH = 1.03 -PEO113-b-PDEA50 and RG/RH = 1.10 - POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47 polyplexes). The structure sensitive parameter provides information on the shape, inner structure and conformation of scattering objects. The structure-sensitive parameter has respectively the values of 0.775, 1.78 and ≥ 2 in the cases of hard-spheres, random coils and rod-like structures.32 Furthermore, the ρ-value of spherical objects is dependent on the inner structure and compactness33 being close to 0.775 for compact spheres, ρ ~ 0.8-0.9 for block copolymer micelles due to solvation phenomena34 and ρ ~ 1.0 for hollow spheres and vesicles.35 The determined values suggest that the supramolecular structures are spherical, however, highly swollen by water. The surface morphology of the produced polyplexes has been in parallel probed by atomic force microscopy (AFM). The images suggest that POEGMA70-bP(OEGMA10-co-DEA47-co-DPA47)/ctDNA polyplexes are reasonable homogeneous in size and they possess a nearly spherical or slightly elongated morphology. The drying process inevitably eases the sticking of particles to some extent as can be seen in Figure 6F. The PEO113-b-PDEA50/ctDNA polyplexes seems to hold a more irregular


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morphology and the observed population is more heterogeneous (Figure 6C). Additionally, PEO113-b-PDEA50/ctDNA polyplexes adopt a more “hairy” structure. The surface differences are probably related to distinct chemistry of the polymer stabilizing shells (POEGMA; PEO) and/or adopted polymer conformations. (A)

12

10 1 / Isc(q) (a.u.)

1 / Isc(q) (a.u.)

(D)

12

10

8

8

6

6

4

4

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600

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(E)

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0.6

0.6 C (t)

C (t)

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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0.4

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F

Figure 6. Static light scattering (1/Isc vs. q2), autocorrelation functions measured at 90o with respective distribution of relaxation times and AFM images for PEO113-b-PDEA50 (A-C) and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) (D-F) polyplexes at N/P = 2.0.


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The size of the supramolecular aggregates are in reasonable agreement with the light scattering data (DH = 2RH ~ 200 nm). Discrepancies are indeed expected since the dehydration caused by solvent evaporation during casting may reduce their sizes. On the other way around, the softness of the nanostructures may allow spreading onto the mica surface which may then cause also overestimation of sizes. These detailed characterizations have been also complemented by small-angle X-ray scattering measurements in order to shed more light onto the inner structural features of the supramolecular aggregates. In such systems, the low-q range probes the large aggregates whereas the high-q range is essentially related to the ctDNA chain due to its higher electron density contrast. The scattering patterns of PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) polyplexes along to the pattern of pure ctDNA are given in Figure S10. It is evidenced at the low-q range an upward profile of the scattering intensity. The scattering intensity of pure ctDNA follows a q-1.1 which corresponds to a roughly cylindrical structure. The power law of the supramolecular aggregates evidences that the structures are not cylindrical and they do not hold a defined sharp interface (q-4). The determined values (q-2.6 and q-3.5) instead suggest a reasonable compact fractal network although without a particular structuring and/or shape. Additionally, as the power law of POEGMA70-b-P(OEGMA10-co-DEA47co-DPA47)/ctDNA polyplexes is closer to q-4 it suggests a more compacted as compared to a more loosely packed structure of PEO113-b-PDEA50 polyplexes. The results are indeed in fully agreement with the SDLS data and the RG/RH ratios determined, as well as with the AFM observations.

3.4

Thermodynamics of Polyplexes Formation


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Isothermal titration calorimetry (ITC) was performed to obtain the thermodynamic data related to the polymer-DNA binding processes. The ITC raw data for titration of ctDNA with PEO113-b-PDPA50, PEO113-b-PDEA50, POEGMA70-bP(OEGMA10-co-DAMA44) and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) are given in Figure S11 along with details of the resulting integrated heat (Supporting Information File). The summary of the resulting integrated heat per mol of injectant (N) as a function of N/P and the fitting results (solid lines) are given in Figure 7. 16 14 12 kcal/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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10 8 6 4 2 0 -2 0

1

2

3

4

5

6

N/P

Figure 7. ITC resulting integrated heat per mol of injectant (N) as a function of N/P along with the fitting results (solid lines) for titration of ctDNA with PEO113-b-PDPA50 (○),

POEGMA70-b-P(OEGMA10-co-DAMA44)

(▲),

PEO113-b-PDEA50 (■)

and

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

At a glance, looking at PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-coDEA47-co-DPA47), the ITC data evidenced that interactions gave rise to endothermic binding signals. On the other hand, taking into account PEO113-b-PDPA50, there can be seen exothermic binding signals. Nevertheless, its inability to produce ctDNA polyplexes is again confirmed as the heat signals are small. Similar behavior was obtained in the titration of ctDNA with POEGMA70-b-P(OEGMA10-co-DAMA44). In such a case, the thermodynamic data could not even be fitted due to negligible energy


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outputs. Apart from POEGMA70-b-P(OEGMA10-co-DAMA44), the heat profiles could be adequately fitted by using the one-site model according to Equation S5 (Supporting Information File). It can be visually seen that the theoretical curves represent properly the experimental profiles in Figure 7. Additionally, this is supported quantitatively by the residual plots (Figure S12). Although the complexity of the systems suggests multisite interaction, the adequate fittings achieved by using the one-site model evidence that complexation occurs essentially by one-site of high affinity which allowed the extraction of single values of K, N and ∆H. Therefore, quantitative thermodynamic parameters of weaker binding sites are probably overwhelmed by the thermodynamic outputs of the site with highest affinity. The extracted thermodynamic parameters are given in Table 1. Table 1. Thermodynamics and binding parameters obtained from ITC data.

Parameter

PEO113-bPDPA

PEO113-bPDEA50

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

N K (104M-1) ∆H (kcal.mol-1) ∆G (kcal.mol-1) ∆S (cal.mol-1K-1)

1.2 2.6 -0.8 -6.0 17

5.0 0.4 1.2 -4.9 21

1.4 9.0 19 -6.8 87

The determined binding constants are in the range 103-105 M-1 which agree with reported values for cationic polymers.36–38 The highest binding affinity was determined for POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47). Similarly, the enthalpy of binding is substantially larger for POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) as compared to PEO113-b-PDEA50. The enthalpic binding term is generally the result of a combination of electrostatics, conformational changes and hydrogen bonding interactions and the magnitude cannot simply be related to one contribution. Nevertheless, it is well accepted that polyelectrolyte associations are mainly


Page 25 of 35

entropically driven processes.39,40 Thus, endothermic signals recorded during polyplexes formation are typically present. Interestingly, the polymer POEGMA70-b-P(OEGMA10co-DEA47-co-DPA47), which gave rise to the highest degree of DNA condensation according to the previously reported FLCS results, is energetically the least favorable process (more endothermic). Understand these variations is challenging since it is daunting to decode all the contributions to the heat factor as many energetic components are occurring concomitantly during the association. Nevertheless, the unfavorable enthalpy of binding is balanced by the highest entropy variation (87 cal.mol-1.K-1). Indeed, all the processes are spontaneous and entropically driven as given by the data reported in Table 1. The entropy increase is mainly due to the release of solvent molecules and release of monovalent counterions from the DNA phosphate groups upon attraction.41

3.5

DNA Conformation upon Compaction Circular dichroism and UV spectroscopy were performed to verify possible

DNA conformational changes induced by the condensation. The Figure 8 portrays the CD signal of ctDNA and polyplexes produced from PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) at N/P = 2.0. 9 CD Ellipticity (millidegrees)

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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6 3 0 -3 -6 -9 240

260

280

300

320

wavelenght (nm)

Figure 8. CD spectra of pure ctDNA (●) and polyplexes produced from PEO113-bPDEA50 (○) and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) (■) at N/P = 2.0.


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The polycations do not exhibit any absorbance band in the region and the CD spectra display ctDNA molar ellipticity only. The reported spectra is characteristic of a duplex in B conformation exhibiting a negative peak at ~ 248 nm and a positive peak at ~ 274 nm corresponding to the π⟶π* transition of the nucleotide bases.42 The data clearly indicate that the conformation of ctDNA remained the same, as the spectra in the presence of either PEO113-b-PDEA50 or POEGMA70-b-P(OEGMA10-co-DEA47-coDPA47) are very similar to the spectra of pure ctDNA. The complexation therefore seems not to affect the secondary structure of the biological entity. The result also suggests that the complexation is mainly attributed to electrostatic forces since strong H-boding interaction would alter the CD profile.43 Correspondingly, changes in the maximum of the UV spectra of ctDNA in the presence of the polycations were not seen, however, a reduction in the UV absorption as a function of N/P was clearly perceptible (Supporting Information File - Figure S13). This suggests a reduction of free ctDNA in solution as the polycations are progressively added to the system.

3.6

Stability of the Polyplexes The stability of the produced polyplexes has been probed in serum environment.

The serum stability is one of the criteria for their usefulness as gene carriers. The serum albumin is the most abundant plasma protein in mammals. Herein, bovine serum albumin (BSA) which is a large globular protein (Mw = 66.5 kDa) was used as the model serum protein. It corresponds to about half of the blood serum proteins and the value for albumin concentration in mammals is about 40 mg.mL-1.44 The isoelectric point of BSA is 4.7-4.945 and consequently, it is slightly negatively charged in the healthy physiological environment (pH 7.4). The distribution of RH for PEO113-b-PDEA50 and


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POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) polyplexes (N/P = 2.0) in presence of BSA (40.0 mg.mL-1) at t = 0h (○) and t = 8h (□) are shown in Figure 9. The distributions in BSA environment are bimodal. The population centred at RH ~ 4 nm is related to the presence of free BSA. One can visually notice a slight increase in the size of the supramolecular aggregates in BSA environment. The size increased from ~ 72 nm to ~ 87 nm and from ~ 85 nm to ~100 nm for PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47)

polyplexes

respectively.

This

behavior is certainly related to the adsorption of a protein layer onto de surface of the supramolecular aggregates. Nevertheless, the assemblies remain stable over the investigated period. (A)

1.0

Amplitude (a.u)

0.8

0.6

0.4

0.2

0.0 0.1

1

10

100

1000

10000

RH (nm) 1.0

(B)

0.8 Amplitude (a.u)

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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0.6

0.4

0.2

0.0 0.1

1

10

100

1000

10000

RH (nm)

Figure 9. Distribution of RH for PEO113-b-PDEA50 (A) and POEGMA70-bP(OEGMA10-co-DEA47-co-DPA47) (B) polyplexes at N/P = 2.0 in BSA-free (○) and in the presence of BSA 40.0 mg.mL-1 after 8 hs - incubation time (□).


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This finding is of due importance as a number of potentially promising gene delivery systems failed in such property.5,46 The nearly neutral ζ-potential of the assemblies and therefore the steric shielding effect of PEO or POEGMA chains are supposed to be the responsible for such outstanding stability. The formation of a protein layer onto the surface of stable assemblies in serum environment is indeed well accepted nowadays and it is what the cells will see to the end.47 The stability in plasma environment was also confirmed (Figure S14).

4. Conclusions Herein, it has been demonstrated the ability of PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) to properly condense ctDNA in an entropically driven process. The produced polyplexes are about 70-80 nm and therefore with a desired size for cellular uptake via endocytic pathways. The supramolecular assemblies are highly swollen by water and the complete set of experimental data evidenced that POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) condenses ctDNA more efficiently and with higher thermodynamic outputs as compared to PEO113-bPDEA50. The circular dichroism data indicated that the conformation of ctDNA remained the same after complexation and the polyplexes are highly stable in serum environment. The biological assays (cytotoxicity, cellular uptake and in-vitro cell transfection) are currently underway aiming to identify especially if the polyplexes produced from POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) fulfill the further requirements (endosome-disrupting ability, efficient intracellular DNA release and low toxicity) for an efficient gene transfection vector. The investigations may guide the novel block copolymer to the class of potential soft materials towards effective gene therapies.


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Associated Content Details of characterization procedures and auxiliary experimental data (ITC, UV-vis, light scattering) are given as Supporting Information. The Supporting

Information File is available free of charge on the ACS Publications website.

Acknowledgements This work has been sponsored by FAPESP (Grant No. 2014/22983-9), CNPq (Grant No. 470608/2012-9) and by the Ministry of Education, Youth and Sports of the Czech Republic (Grant No. LH14292). F.C.G thanks the productivity research fellowship granted by CNPq. The CEM at UFABC is acknowledged for the accessibility to the Malvern light scattering equipment. The LNLS is acknowledged for supplying the SAXS beam time (proposal 12536).

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