Complexation behavior of PNIPAM-b-QPDMAEA copolymer aggregates with linear DNAs of different lengths
Graphical abstract
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 NN,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)
- et al.
Micelleplexes as nucleic acid delivery systems for cancer-targeted therapies
J. Controlled Release
(2020) - et al.
Development of RNAi technology for targeted therapy—a track of siRNA based agents to RNAi therapeutics
J. Controlled Release
(2014) - et al.
Functional lipids and lipoplexes for improved gene delivery
Biochimie
(2012) - et al.
Temperature-responsive cationic block copolymers as nanocarriers for gene delivery
Int. J. Pharm.
(2013) - et al.
Recent progress in gene delivery using non-viral transfer complexes
J. Controlled Release.
(2001) - et al.
Poly(ethylemine) ans its role in gene delivery
J. Controlled Release.
(1999) - et al.
Revisit complexation between DNA and polyethylenimine—effect of uncomplexed chains free in the solution mixture on gene transfection
J. Controlled Release.
(2011) - et al.
Nanotechnological approaches for the delivery of macromolecules
J. Controlled Release.
(2003) - et al.
PDMAEMA based gene delivery materials
Mater. Today
(2012) - et al.
A direct comparison of linear and star-shaped poly (dimethylaminoethyl acrylate) polymers for polyplexation with DNA and cytotoxicity in cultured cell lines
Eur. Polym. J.
(2017)
Poly (N-isopropylacrylamide)(PNIPAM) is never hydrophobic
J. Colloid Interface Sci.
The biological stimuli for governing the phase transition temperature of the “smart” polymer PNIPAM in water, Colloids Surf
B.
The gene transfection efficiency of thermoresponsive N, N, N-trimethyl chitosan chloride-g-poly (N-isopropylacrylamide) copolymer
Biomaterials
In vitro transfection of HeLa cells with temperature sensitive polycationic copolymers
J. Controlled Release.
Gene therapy for nucleus pulposus regeneration by heme oxygenase-1 plasmid DNA carried by mixed polyplex micelles with thermo-responsive heterogeneous coronas
Biomaterials
Temperature-responsive cationic block copolymers as nanocarriers for gene delivery
Int. J. Pharm.
PNIPAM-b-PDMAEA double stimuli responsive copolymers: Effects of composition, end groups and chemical modification on solution self-assembly
Eur. Polym. J.
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.
Hydrophilic/hydrophobic modifications of a PnBA-b-PDMAEA copolymer and complexation behaviour with short DNA
Eur. Polym. J.
Assembly strategy of liposome and polymer systems for siRNA delivery
Int. J. Pharm.
Nonviral vectors for gene delivery
Chem. Rev.
Design and development of polymers for gene delivery
Nat. Rev. Drug Discov.
Adeno-associated virus vector as a platform for gene therapy delivery
Nat. Rev. Drug Discov.
Progress and problems with the use of viral vectors for gene therapy
Nat. Rev. Genet.
Intelligent polymers as nonviral vectors
Gene Ther.
Development of polymeric micelles for targeting intractable cancers
Cancer Sci.
Block copolymer micelles in nanomedicine applications
Chem. Rev.
Gene delivery by lipoplexes and polyplexes
Cited by (5)
Structure of micelleplexes formed between QPDMAEMA-b-PLMA amphiphilic cationic copolymer micelles and DNA of different lengths
2022, European Polymer JournalCitation Excerpt :The binding affinity of QPDMAEMA-b-PLMA copolymer micelles was examined by fluorescence spectroscopy using ethidium bromide quenching assay. Ethidium bromide (EtBr) is a cationic fluorescent dye that can interact with DNA by intercalation into the base pairs of the double helix [52–54]. EtBr intercalated into the DNA helix (DNA-EtBr) exhibits strong fluorescence intensity.
Aggregation behavior of triblock terpolymer poly[n-butyl acrylate-block-N-isopropylacrylamide-block-2-(dimethylamino)ethyl acrylate] at the air/water interface
2022, Journal of Applied Polymer Science