Complexation behavior of PNIPAM-b-QPDMAEA copolymer aggregates with linear DNAs of different lengths

https://doi.org/10.1016/j.eurpolymj.2021.110575Get rights and content

Highlights

  • HOOC-PNIPAM-b-QPDMAEA-C12H25 thermoresponsive-cationic diblock copolymers form polyplexes with linear DNAs.

  • Complexation is confirmed by fluorescence measurements of ethidium bromide quenching.

  • The length of DNA, the block copolymer composition, and the N/P ratio control the structure of polyplexes.

  • Polyplexes with increased size and surface charge are formed at temperatures above the LCST of PNIPAM block.

Abstract

We investigate the complexation ability of CH3I quaternized HOOC-poly(N-isopropylacrylamide)-b-poly(2-(dimethylamino) acrylate)-C12H25 (HOOC-PNIPAM-b-QPDMAEA-C12H25) thermoresponsive-cationic diblock copolymers, having different composition of the blocks, with linear DNA molecules of different lengths. The thermoresponsive-cationic diblock copolymers are able to self-assemble into nanosized aggregates in aqueous media, where the PNIPAM block possesses the inner part and the QPDMAEA block constitutes the outer part, even at ambient temperature (temperature below the LCST value of PNIPAM block) because of the presence of hydrophobic C12H25 groups placed at the QPDMAEA free chain end. Thermoresponive-cationic copolymer/DNA polyplexes were prepared at various N/P (amine over phosphate groups) ratios utilizing two DNAs of different lengths (DNAshort ≈ 113 bp and DNAlong ≈ 2000 bp). Fluorescence measurements of ethidium bromide quenching as well as UV–vis measurements reveal the complexation of the cationic polymeric aggregates with DNA molecules. In addition, dynamic and electrophoretic light scattering measurements show the structural features and the surface charge of the formed polyplexes at both 25 °C and 45 °C (temperature above the LCST value of PNIPAM block). The salt tolerance of the formed polyplexes was also examined by dynamic light measurements. The overall physiochemical characterization of the polyplexes provides new insights into the parameters affecting the interactions between thermoresponsive-cationic polymer aggregates and nucleic acids.

Introduction

Over the Past decades, the scientific interest has been focused on gene therapy as an alternative therapeutic approach because it presents considerable advantages compared to conventional treatment modalities. [1] Gene therapy has demonstrated tremendous potential in the treatment of several genetic and acquired diseases through the targeted delivery of genetic material to the infected cells and tissues. [2] The utilization of a gene vector with appropriate features is of great significance for the successful delivery of nucleic acids into the cells. In particular, an efficient gene carrier should be capable of condensing the large molecules of DNA (deoxyribonucleic acid) to suitable sizes for cellular uptake and concurrently protecting them from extra- and intracellular nuclease degradation. Furthermore, the carrier must be nontoxic, nonimmunogenic and nonpathogenic so as to deliver nucleic acids to the affected cells not only efficiently but also safely [3], [4], [5], [6].

At the beginning, the research was principally concentrated on the use of viruses as viral gene vectors due to their capability of overcoming the extra- and intracellular barriers and offering high gene delivery efficacy [5]. However, viral gene carriers display significant disadvantages, specifically, immunogenicity potential, possibility of mutagenesis, broad tropism, obstacles to production on a large scale and insufficient capacity so as to carry, protect and deliver large nucleic acid molecules. [7] For the aforementioned reasons, nonviral gene delivery vectors have attracted increased scientific interest since they display significant advantages, including their production on a large scale in a more facile way, their lower cost and their reduced immunogenic response in several cases [2], [3], [8].

Among nonviral gene vectors, cationic polymers [9], [10], [11], [12] and lipids [13], [14], [15] have emerged as efficient carriers in gene therapy strategies. However, the utilization of cationic polymers as gene vectors demonstrates some advantages in comparison to the utilization of lipids, namely the development of polyplexes with relatively small size and narrow distributions, the high stability of the polyplexes against nuclease degradation and the prospect of increasing the payload of the carriers [16]. The synthesis of cationic polymers with controlled molecular weights, compositions and architecture are parameters of great significance in designing and developing efficient gene delivery systems. Cationic polymers display the tendency to form polyplexes after simple mixing with DNA molecules due to the electrostatic interactions between the positively charged groups derived from the cationic groups of the polymer and the negatively charged phosphate groups of DNA [17].

Cationic polymers such as poly(l-lysine) (PLL) [18], [19], [20], [21], polyethylenemine (PEI) [22], [23] and poly(2-(dimethylamino)ethyl methacrylate) PDMAEMA [24], [25], [26], [27], have been investigated intensively as nonviral gene vectors, exhibiting high gene transfection efficiency. However, in some cases PDMAEMA-based polymers present toxicity, setting limitations to their application. In recent years, the acrylate version of PDMAEMA polymer, the poly(2-(dimethylamino)ethyl acrylate) (PDMAEA)-based polymers have received increasing attention due to several considerable advantages including lack of immunogenicity, low cytotoxicity in cultured cells [28] and sufficient loading capacity [29].

Stimuli-responsive polymers constitute fascinating gene carriers, since the interaction with DNA molecules can be altered in response to small variations in the external environment. One of the most well-known paradigm of these so-called “intelligent’’ polymers, is the temperature-responsive poly-(N-isopropylacrylamide) (PNIPAM), which demonstrates hydrophilic character in aqueous media at low temperatures and becomes less hydrophilic as the temperature increases above its lower critical solution temperature value (LCST). The LCST value of PNIPAM polymer is around 32 °C, close to physiological body temperature, that renders it an extremely appealing polymer for gene delivery applications. [30], [31], [32], [33], [34], [35] Several PNIPAM copolymers have been extensively researched in the pursuit of novel nonviral carriers for gene therapy strategies, such as Nsingle bondN,N-trimethyl chitosan chloride-g-(N-isopropylacrylamide) [36], poly[2-(dimethylamino)ethyl methacrylate-co-[cisbutenedioic anhydride-poly[(N-isopropylacrylamide)–co-(butylmethacrylate)]]] [37], poly[(N-isopropylacrylamide-b-ethyleneimine [38], [39] and poly(N-isopropylacrylamide)-block-PAsp(DET) [40]. Conformationally collapsed PNIPAM chains at the periphery of polyplexes can protect better DNA cargo from enzymatic degradation, due to the increased density of the thermoresponsive corona segments at physiological temperatures above the LCST, and may increase polyplex interaction with cell membranes, due to increased hydrophobicity without total dehydration, thus enhancing cellular uptake of polyplexes and enhance their transfection efficiency [41], [42], [43].

In the present work, the ability of HOOC-PNIPAM-b-QPDMAEA-C12H25 thermoresponsive-cationic aggregates to interact with DNA molecules of different lengths is investigated. Two DNA samples, one short-113 bp and one long-2000 bp from salmon testes, were used in order to study the effects of DNA length on the complexes formed. For this purpose, two diblock copolymers synthesized in our previous study [43] having different composition of the blocks were utilized. These diblock copolymers are constituted by one temperature-responsive PNIPAM block and one polyelectrolyte QPDMAEA block and self-assemble into nanoscaled aggregates in aqueous media even at ambient temperature owing to the existence of hydrophobic C12H25 functional groups placed at the QPDMAEA free chain end. The QPDMAEA block carries positive charges on its repeating units capable of binding with oppositely charged DNA molecules through electrostatic interactions. The main goal of this study is the physiochemical characterization of the formed HOOC-PNIPAM-b-QPDMAEA-C12H25/DNA polyplexes so as to confirm the successful complexation and thereafter to elucidate the parameters that may affect polyplex structure and stability at different temperatures.

Section snippets

Materials

Deoxyribonucleic acid sodium salt, from salmon testes, DNAShort ≈ 113 bp was obtained from Sigma-Aldrich and DNAlong ≈ 2000 bp was purchased from Acros. Ethidium bromide (EtBr) utilized as the fluorescent DNA intercalating probe for assays of polyplexes formation as well as sodium chloride (NaCl, ≥99.0%) were received from Sigma-Aldrich and used as obtained without further purification.

Synthesis and self-assembly of diblock copolymers

HOOC-PNIPAM52-b-QPDMAEA15-C12H25-1 and HOOC-PNIPAM33-b-QPDMAEA23-C12H25-2 thermoresponsive-cationic block

Results and discussion

Studies on the electrostatic interactions between polymers containing cationic moieties and nucleic acids indisputably facilitate the clarification of DNA complexation features, packaging and delivery that are strongly connected to gene transfection efficacy. The complexation of cationic polymers with nucleic acids and particularly, the formation of nanosized polyplexes is a complicated procedure, influenced by a variety of parameters such as polymer chain length [45], N/P ratio [46], solution

Conclusions

In this study, the cationic and thermoresponsive aggregates of HOOC-PNIPAM-b-QPDMAEA-C12H25 diblock copolymers, having different composition of the blocks were found to bind with nucleic acids molecules through electrostatic interactions and form polyplexes, presenting colloidal stability. The formation of polyplexes is confirmed by FS measurements of ethidium bromide quenching, as well as UV–vis measurements. In particular, the extracted results reveal a strong interaction between cationic

Data Availability

Data will be available upon request.

CRediT authorship contribution statement

Despoina Giaouzi: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing - original draft. Stergios Pispas: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Visualization, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research is co-financed by Greece and the European Union (European Social Fund-ESF) through the Operational Programme “Human Resources Development, Education and Lifelong Learning” in the context of the project “Strengthening Human Resources Research Potential via Doctorate Research – 2 nd Call’’ (MIS-5000432), implemented by the State Scholarships Foundation (ΙΚΥ).

References (57)

  • R. Pelton

    Poly (N-isopropylacrylamide)(PNIPAM) is never hydrophobic

    J. Colloid Interface Sci.

    (2010)
  • R. Umapathi et al.

    The biological stimuli for governing the phase transition temperature of the “smart” polymer PNIPAM in water, Colloids Surf

    B.

    (2015)
  • Z. Mao et al.

    The gene transfection efficiency of thermoresponsive N, N, N-trimethyl chitosan chloride-g-poly (N-isopropylacrylamide) copolymer

    Biomaterials

    (2007)
  • M. Türk et al.

    In vitro transfection of HeLa cells with temperature sensitive polycationic copolymers

    J. Controlled Release.

    (2004)
  • G. Feng et al.

    Gene therapy for nucleus pulposus regeneration by heme oxygenase-1 plasmid DNA carried by mixed polyplex micelles with thermo-responsive heterogeneous coronas

    Biomaterials

    (2015)
  • M.T. Calejo et al.

    Temperature-responsive cationic block copolymers as nanocarriers for gene delivery

    Int. J. Pharm.

    (2013)
  • D. Giaouzi et al.

    PNIPAM-b-PDMAEA double stimuli responsive copolymers: Effects of composition, end groups and chemical modification on solution self-assembly

    Eur. Polym. J.

    (2020)
  • A.J. Geall et al.

    Rapid and sensitive ethidium bromide fluorescence quenching assay of polyamine conjugate–DNA interactions for the analysis of lipoplex formation in gene therapy

    J. Pharm. Biomed. Anal.

    (2000)
  • A. Chroni et al.

    Hydrophilic/hydrophobic modifications of a PnBA-b-PDMAEA copolymer and complexation behaviour with short DNA

    Eur. Polym. J.

    (2020)
  • H. Song et al.

    Assembly strategy of liposome and polymer systems for siRNA delivery

    Int. J. Pharm.

    (2020)
  • M.A. Mintzer et al.

    Nonviral vectors for gene delivery

    Chem. Rev.

    (2009)
  • D.W. Pack et al.

    Design and development of polymers for gene delivery

    Nat. Rev. Drug Discov.

    (2005)
  • D. Wang et al.

    Adeno-associated virus vector as a platform for gene therapy delivery

    Nat. Rev. Drug Discov.

    (2019)
  • C.E. Thomas et al.

    Progress and problems with the use of viral vectors for gene therapy

    Nat. Rev. Genet.

    (2003)
  • S. Dincer et al.

    Intelligent polymers as nonviral vectors

    Gene Ther.

    (2005)
  • N. Nishiyama et al.

    Development of polymeric micelles for targeting intractable cancers

    Cancer Sci.

    (2016)
  • H. Cabral et al.

    Block copolymer micelles in nanomedicine applications

    Chem. Rev.

    (2018)
  • C.T. de Ilarduya et al.

    Gene delivery by lipoplexes and polyplexes

  • Cited by (5)

    View full text