Optical dark-field spectroscopy of single plasmonic nanoparticles for molecular biosciences.

An ideal sensor capable of quantifying analytes in minuscule sample volumes represents a significant technological advancement. Plasmonic nanoparticles integrated with optical dark-field spectroscopy have reached this capability, demonstrating versatility and expanding applicability across in vitro and in vivo subjects. This review underscores the applicability of optical dark-field spectroscopy with single plasmonic nanoparticles to elucidate a wide range of biomolecular characteristics, including binding constants, molecular dynamics, distances, and forces, as well as recording cell communication signals. Perspectives highlight the potential for the development of implantable nanosensors for metabolite detection in animal models, illustrating the technique's efficacy without the need for labeling molecules. In summary, this review aims to consolidate knowledge of this adaptable and robust technique for decoding molecular biological phenomena within the nano- and bio-scientific community.

Effects of swift heavy ions on metal nanoparticles embedded in silica: Using linearly polarized light to monitor the elongation kinetics.

The elongation of metallic nanoparticles (NPs) embedded in a dielectric matrix after irradiation with swift heavy ions is a phenomenon that has been known for several years. However, the precise mechanism behind this deformation process is still not fully understood, primarily due to the dearth of information during intermediate stages of deformation. In this study, we report the continuation of our previous work [Peña-Rodríguez et al., Sci. Rep. 7(1), 922 (2017)], exploiting the strong dependence of the localized surface plasmon resonance on the aspect ratio of elongated metal NPs to study the elongation kinetics in situ. In situ optical absorption spectra were measured using a polarizing beam splitter to separate the longitudinal and transverse plasmon modes of the anisotropic NPs. Then, the detailed geometrical and compositional parameters were determined from a fit of these spectra. The use of linearly polarized light allowed for a more accurate analysis of the elongation kinetics, particularly useful in the first stages, where longitudinal and transverse modes overlap.

From Multi- to Single-Hollow Trimetallic Nanocrystals by Ultrafast Heating.

Metal nanocrystals (NCs) display unique physicochemical features that are highly dependent on nanoparticle dimensions, anisotropy, structure, and composition. The development of synthesis methodologies that allow us to tune such parameters finely emerges as crucial for the application of metal NCs in catalysis, optical materials, or biomedicine. Here, we describe a synthetic methodology to fabricate hollow multimetallic heterostructures using a combination of seed-mediated growth routes and femtosecond-pulsed laser irradiation. The envisaged methodology relies on the coreduction of Ag and Pd ions on gold nanorods (Au NRs) to form Au@PdAg core-shell nanostructures containing small cavities at the Au-PdAg interface. The excitation of Au@PdAg NRs with low fluence femtosecond pulses was employed to induce the coalescence and growth of large cavities, forming multihollow anisotropic Au@PdAg nanostructures. Moreover, single-hollow alloy AuPdAg could be achieved in high yield by increasing the irradiation energy. Advanced electron microscopy techniques, energy-dispersive X-ray spectroscopy (EDX) tomography, X-ray absorption near-edge structure (XANES) spectroscopy, and finite differences in the time domain (FDTD) simulations allowed us to characterize the morphology, structure, and elemental distribution of the irradiated NCs in detail. The ability of the reported synthesis route to fabricate multimetallic NCs with unprecedented hollow nanostructures offers attractive prospects for the fabrication of tailored high-entropy alloy nanoparticles.

Tunable gold nanorod/NAO conjugates for selective drug delivery in mitochondria-targeted cancer therapy.

Nonyl acridine orange (NAO) is a lipophilic and positively charged molecule widely used as a mitochondrial fluorescent probe. NAO is cytotoxic at micromolar concentration and might be potentially used as a mitochondria-targeted drug for cancer therapy. However, the use of NAO under in vivo conditions would be compromised by the unspecific interactions with off-target cells and negatively charged proteins present in the bloodstream. To tackle this limitation, we have synthesized NAO analogues carrying an imidazole group for their specific binding to nitrilotriacetic (NTA) functionalized gold nanorods (AuNRs). We demonstrate that AuNRs provide 104 binding sites and a controlled delivery under acidic conditions. Upon incubation with mouse embryonic fibroblasts, the endosomal acidic environment releases the NAO analogues from AuNRs, as visualized through the staining of the mitochondrial network. The addition of the monoclonal antibody Cetuximab to the conjugates enhanced their uptake within lung cancer cells and the conjugates were cytotoxic at subnanomolar concentrations (c50 ≈ 0.06 nM). Moreover, the specific interactions of Cetuximab with the epidermal growth factor receptor (EGFR) provided a specific targeting of EGFR-expressing lung cancer cells. After intravenous administration in patient-derived xenografts (PDX) mouse models, the conjugates reduced the progression of EGFR-positive tumors. Overall, the NAO-AuNRs provide a promising strategy to realize membrane mitochondria-targeted conjugates for lung cancer therapy.

Transgene expression in mice of the Opa1 mitochondrial transmembrane protein through bicontinuous cubic lipoplexes containing gemini imidazolium surfactants.

BACKGROUND: Lipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, also known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. The polar head can carry a large variety of chemical groups including amines as well as guanidino or imidazole groups. In particular, gemini cationic lipids consist of two positive polar heads linked by a spacer with different length. As for the hydrophobic aliphatic chains, they can be unsaturated or saturated and are connected to the polar head-groups. Many other chemical components can be included in the formulation of lipoplexes to improve their transfection efficiency, which often relies on their structural features. Varying these components can drastically change the arrangement of DNA molecules within the lamellar, hexagonal or cubic phases that are provided by the lipid matrix. Lipofection is widely used to deliver genetic material in cell culture experiments but the simpler formulations exhibit major drawbacks related to low transfection, low specificity, low circulation half-life and toxicity when scaled up to in vivo experiments. RESULTS: So far, we have explored in cell cultures the transfection ability of lipoplexes based on gemini cationic lipids that consist of two C16 alkyl chains and two imidazolium polar head-groups linked with a polyoxyethylene spacer, (C16Im)2(C4O). Here, PEGylated lipids have been introduced to the lipoplex formulation and the transgene expression of the Opa1 mitochondrial transmembrane protein in mice was assessed. The addition of PEG on the surface of the lipid mixed resulted in the formation of Ia3d bicontinuous cubic phases as determined by small angle X-ray scattering. After a single intramuscular administration, the cubic lipoplexes were accumulated in tissues with tight endothelial barriers such as brain, heart, and lungs for at least 48 h. The transgene expression of Opa1 in those organs was identified by western blotting or RNA expression analysis through quantitative polymerase chain reaction. CONCLUSIONS: The expression reported here is sufficient in magnitude, duration and toxicity to consolidate the bicontinuous cubic structures formed by (C16Im)2(C4O)-based lipoplexes as valuable therapeutic agents in the field of gene delivery.

Controlled pDNA Release in Gemini Cationic Lipoplexes by Femtosecond Laser Irradiation of Gold Nanostars.

The design of nanovectors able to overcome biological barriers is one of the main challenges in biomedicine. Gemini cationic lipids are considered potential candidates for gene therapy due to their high biocompatibility and capacity to condense nucleic acids safely in the form of lipoplexes. However, this approach presents difficulties regarding genetic unpacking and, therefore, control over this process becomes crucial to ensure successful transfection. In this work, gemini cationic lipoplexes were prepared in the presence of plasmonic gold nanostars (AuNSs) to afford a nanovector that efficiently releases plasmid DNA (pDNA) upon irradiation with near-infrared femtosecond laser pulses. A critical AuNSs concentration of 50 pM and optimized laser power density of 400 mW led to successful pDNA release, whose efficiency could be further improved by increasing the irradiation time. Agarose gel electrophoresis was used to confirm pDNA release. UV-Vis-NIR spectroscopy and transmission electron microscopy studies were performed to monitor changes in the morphology of the AuNSs and lipoplexes after irradiation. From a physicochemical point of view, this study demonstrates that the use of AuNSs combined with gemini cationic lipoplexes allows control over pDNA release under ultrafast laser irradiation.

Gemini Cationic Lipid-Type Nanovectors Suitable for the Transfection of Therapeutic Plasmid DNA Encoding for Pro-Inflammatory Cytokine Interleukin-12.

Ample evidence exists on the role of interleukin-12 (IL-12) in the response against many pathogens, as well as on its remarkable antitumor properties. However, the unexpected toxicity and disappointing results in some clinical trials are prompting the design of new strategies and/or vectors for IL-12 delivery. This study was conceived to further endorse the use of gemini cationic lipids (GCLs) in combination with zwitterionic helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine) as nanovectors for the insertion of plasmid DNA encoding for IL-12 (pCMV-IL12) into cells. Optimal GCL formulations previously reported by us were selected for IL-12-based biophysical experiments. In vitro studies demonstrated efficient pCMV-IL12 transfection by GCLs with comparable or superior cytokine levels than those obtained with commercial control Lipofectamine2000*. Furthermore, the nanovectors did not present significant toxicity, showing high cell viability values. The proteins adsorbed on the nanovector surface were found to be mostly lipoproteins and serum albumin, which are both beneficial to increase the blood circulation time. These outstanding results are accompanied by an initial physicochemical characterization to confirm DNA compaction and protection by the lipid mixture. Although further studies would be necessary, the present GCLs exhibit promising characteristics as candidates for pCMV-IL12 transfection in future in vivo applications.

Insights into colloidal nanoparticle-protein corona interactions for nanomedicine applications.

Colloidal nanoparticles (NPs) have attracted significant attention due to their unique physicochemical properties suitable for diagnosing and treating different human diseases. Nevertheless, the successful implementation of NPs in medicine demands a proper understanding of their interactions with the different proteins found in biological fluids. Once introduced into the body, NPs are covered by a protein corona (PC) that determines the biological behavior of the NPs. The formation of the PC can eventually favor the rapid clearance of the NPs from the body before fulfilling the desired objective or lead to increased cytotoxicity. The PC nature varies as a function of the different repulsive and attractive forces that govern the NP-protein interaction and their colloidal stability. This review focuses on the phenomenon of PC formation on NPs from a physicochemical perspective, aiming to provide a general overview of this critical process. Main issues related to NP toxicity and clearance from the body as a result of protein adsorption are covered, including the most promising strategies to control PC formation and, thereby, ensure the successful application of NPs in nanomedicine.

Protein Expression Knockdown in Cancer Cells Induced by a Gemini Cationic Lipid Nanovector with Histidine-Based Polar Heads.

A histidine-based gemini cationic lipid, which had already demonstrated its efficiency as a plasmid DNA (pDNA) nanocarrier, has been used in this work to transfect a small interfering RNA (siRNA) into cancer cells. In combination with the helper lipid monoolein glycerol (MOG), the cationic lipid was used as an antiGFP-siRNA nanovector in a multidisciplinary study. Initially, a biophysical characterization by zeta potential (ζ) and agarose gel electrophoresis experiments was performed to determine the lipid effective charge and confirm siRNA compaction. The lipoplexes formed were arranged in Lα lamellar lyotropic liquid crystal phases with a cluster-type morphology, as cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS) studies revealed. Additionally, in vitro experiments confirmed the high gene knockdown efficiency of the lipid-based nanovehicle as detected by flow cytometry (FC) and epifluorescence microscopy, even better than that of Lipofectamine2000*, the transfecting reagent commonly used as a positive control. Cytotoxicity assays indicated that the nanovector is non-toxic to cells. Finally, using nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS), apolipoprotein A-I and A-II followed by serum albumin were identified as the proteins with higher affinity for the surface of the lipoplexes. This fact could be beyond the remarkable silencing activity of the histidine-based lipid nanocarrier herein presented.

Biocompatible Nanovector of siRNA Consisting of Arginine-Based Cationic Lipid for Gene Knockdown in Cancer Cells.

Despite the use of small interfering RNAs (siRNAs) as therapeutic agents through the knockdown expression of pathogenic proteins, transportation and delivery of such siRNAs into cells continue to be under investigation. Within nonviral vectors, cationic lipids that include amino acid residues in their structures, and that have already demonstrated their suitability as plasmid DNA nanocarriers, may be also considered as potential siRNA vehicles. A double-chain cationic lipid based on the amino acid arginine mixed with a helper lipid has been the object of this biophysical study. First, ζ-potential measurements and agarose gel electrophoresis experiments confirmed the siRNA compaction, while small-angle X-ray scattering analysis (SAXS) revealed the structural pattern of the lipoplexes. Two bicontinuous cubic phases were found to coexist: the double-gyroid phase (QIIG) and the double-diamond phase (QIID), with Pn3m and Ia3d as crystallographic space groups, respectively; the siRNA is known to be located inside their bicontinuous aqueous channels. Second, in vitro studies in HeLa-green fluorescent protein (GFP) and T731-GFP cell lines (modified for GFP overexpression) showed moderate to high gene knockdown levels (determined by flow cytometry and epifluorescence microscopy) with remarkable cell viabilities (CCK-8 assay). Finally, nano-liquid chromatography/mass spectrometry (nanoLC-MS/MS) was used to identify the nature of the proteins adhered to the surface of the lipoplexes after incubation with human serum, simulating their behavior in biological fluids. The abundant presence of lipoproteins and serum albumin in such protein corona, together with the coexistence of the bicontinuous cubic phases, may be behind the remarkable silencing activity of these lipoplexes. The results reported herein show that the use of amino-acid-based cationic lipids mixed with a suitable helper lipid, which have already provided good results as DNA plasmid nanocarriers in cellular transfection processes, may also be a biocompatible option, and so far little investigated, in gene silencing in vitro strategies.

Au@Ag Core-Shell Nanorods Support Plasmonic Fano Resonances.

In this work, we investigated experimentally and theoretically the plasmonic Fano resonances (FRs) exhibited by core-shell nanorods composed of a gold core and a silver shell (Au@Ag NRs). The colloidal synthesis of these Au@Ag NRs produces nanostructures with rich plasmonic features, of which two different FRs are particularly interesting. The FR with spectral location at higher energies (3.7 eV) originates from the interaction between a plasmonic mode of the nanoparticle and the interband transitions of Au. In contrast, the tunable FR at lower energies (2.92-2.75 eV) is ascribed to the interaction between the dominant transversal LSPR mode of the Ag shell and the transversal plasmon mode of the Au@Ag nanostructure. The unique symmetrical morphology and FRs of these Au@Ag NRs make them promising candidates for plasmonic sensors and metamaterials components.

Intercellular Trafficking of Gold Nanostars in Uveal Melanoma Cells for Plasmonic Photothermal Therapy.

Efficient plasmonic photothermal therapies (PPTTs) using non-harmful pulse laser irradiation at the near-infrared (NIR) are a highly sought goal in nanomedicine. These therapies rely on the use of plasmonic nanostructures to kill cancer cells while minimizing the applied laser power density. Cancer cells have an unsettled capacity to uptake, retain, release, and re-uptake gold nanoparticles, thus offering enormous versatility for research. In this work, we have studied such cell capabilities for nanoparticle trafficking and its impact on the effect of photothermal treatments. As our model system, we chose uveal (eye) melanoma cells, since laser-assisted eye surgery is routinely used to treat glaucoma and cataracts, or vision correction in refractive surgery. As nanostructure, we selected gold nanostars (Au NSs) due to their high photothermal efficiency at the near-infrared (NIR) region of the electromagnetic spectrum. We first investigated the photothermal effect on the basis of the dilution of Au NSs induced by cell division. Using this approach, we obtained high PPTT efficiency after several cell division cycles at an initial low Au NS concentration (pM regime). Subsequently, we evaluated the photothermal effect on account of cell division upon mixing Au NS-loaded and non-loaded cells. Upon such mixing, we observed trafficking of Au NSs between loaded and non-loaded cells, thus achieving effective PPTT after several division cycles under low irradiation conditions (below the maximum permissible exposure threshold of skin). Our study reveals the ability of uveal melanoma cells to release and re-uptake Au NSs that maintain their plasmonic photothermal properties throughout several cell division cycles and re-uptake. This approach may be readily extrapolated to real tissue and even to treat in situ the eye tumor itself. We believe that our method can potentially be used as co-therapy to disperse plasmonic gold nanostructures across affected tissues, thus increasing the effectiveness of classic PPTT.

A Non-Viral Plasmid DNA Delivery System Consisting on a Lysine-Derived Cationic Lipid Mixed with a Fusogenic Lipid.

The insertion of biocompatible amino acid moieties in non-viral gene nanocarriers is an attractive approach that has been recently gaining interest. In this work, a cationic lipid, consisting of a lysine-derived moiety linked to a C12 chain (LYCl) was combined with a common fusogenic helper lipid (DOPE) and evaluated as a potential vehicle to transfect two plasmid DNAs (encoding green fluorescent protein GFP and luciferase) into COS-7 cells. A multidisciplinary approach has been followed: (i) biophysical characterization based on zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and cryo-transmission electronic microscopy (cryo-TEM); (ii) biological studies by fluorescence assisted cell sorting (FACS), luminometry, and cytotoxicity experiments; and (iii) a computational study of the formation of lipid bilayers and their subsequent stabilization with DNA. The results indicate that LYCl/DOPE nanocarriers are capable of compacting the pDNAs and protecting them efficiently against DNase I degradation, by forming Lα lyotropic liquid crystal phases, with an average size of ~200 nm and low polydispersity that facilitate the cellular uptake process. The computational results confirmed that the LYCl/DOPE lipid bilayers are stable and also capable of stabilizing DNA fragments via lipoplex formation, with dimensions consistent with experimental values. The optimum formulations (found at 20% of LYCl content) were able to complete the transfection process efficiently and with high cell viabilities, even improving the outcomes of the positive control Lipo2000*.

Gemini-Based Lipoplexes Complement the Mitochondrial Phenotype in MFN1-Knockout Mouse Embryonic Fibroblasts.

Mitochondria form a dynamic network of constantly dividing and fusing organelles. The balance between these antagonistic processes is crucial for normal cellular function and requires the action of specialized proteins. The mitochondrial membrane proteins mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) are responsible for the fusion of the outer membrane of adjacent mitochondria. Mutations within Mfn1 or Mfn2 impair mitochondrial fusion and lead to some severe mitochondrial dysfunctions and mitochondrial diseases (MDs). A characteristic phenotype of cells carrying defective Mfn1 or Mfn2 is the presence of a highly fragmented mitochondrial network. Here, we use a biocompatible mixture of lipids, consisting on synthetic gemini cationic lipids (GCLs) and the zwitterionic phospholipid (DOPE), to complex, transport, and deliver intact copies of MFN1 gene into MFN1-Knockout mouse embryonic fibroblasts (MFN1-KO MEFs). We demonstrate that the GCL/DOPE-DNA lipoplexes are able to introduce the intact MFN1 gene into the cells and ectopically produce functional Mfn1. A four-fold increase of the Mfn1 levels is necessary to revert the MFN1-KO phenotype and to partially restore a mitochondrial network. This phenotype complementation was correlated with the transfection of GCL/DOPE-MFN1 lipoplexes that exhibited a high proportion of highly packaged hexagonal phase. GCL/DOPE-DNA lipoplexes are formulated as efficient therapeutic agents against MDs.

A Gemini Cationic Lipid with Histidine Residues as a Novel Lipid-Based Gene Nanocarrier: A Biophysical and Biochemical Study.

This work reports the synthesis of a novel gemini cationic lipid that incorporates two histidine-type head groups (C3(C16His)2). Mixed with a helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine (DOPE), it was used to transfect three different types of plasmid DNA: one encoding the green fluorescence protein (pEGFP-C3), one encoding a luciferase (pCMV-Luc), and a therapeutic anti-tumoral agent encoding interleukin-12 (pCMV-IL12). Complementary biophysical experiments (zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and fluorescence anisotropy) and biological studies (FACS, luminometry, and cytotoxicity) of these C3(C16His)2/DOPE-pDNA lipoplexes provided vast insight into their outcomes as gene carriers. They were found to efficiently compact and protect pDNA against DNase I degradation by forming nanoaggregates of 120⁻290 nm in size, which were further characterized as very fluidic lamellar structures based in a sandwich-type phase, with alternating layers of mixed lipids and an aqueous monolayer where the pDNA and counterions are located. The optimum formulations of these nanoaggregates were able to transfect the pDNAs into COS-7 and HeLa cells with high cell viability, comparable or superior to that of the standard Lipo2000*. The vast amount of information collected from the in vitro studies points to this histidine-based lipid nanocarrier as a potentially interesting candidate for future in vivo studies investigating specific gene therapies.

Multidisciplinary Approach to the Transfection of Plasmid DNA by a Nonviral Nanocarrier Based on a Gemini-Bolaamphiphilic Hybrid Lipid.

A multidisciplinary strategy, including both biochemical and biophysical studies, was proposed here to evaluate the potential of lipid nanoaggregates consisting of a mixture of a gemini-bolaamphiphilic lipid (C6C22C6) and the well-known helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) to transfect plasmid DNA into living cells in an efficient and safe way. For that purpose, several experimental techniques were employed, such as zeta potential (phase analysis light scattering methodology), agarose gel electrophoresis (pDNA compaction and pDNA protection assays), small-angle X-ray scattering, cryo-transmission electron microscopy, atomic force microscopy, fluorescence-assisted cell sorting, luminometry, and cytotoxicity assays. The results revealed that the cationic lipid and plasmid offer only 70 and 30% of their nominal positive () and negative charges (), respectively. Upon mixing with DOPE, they form lipoplexes that self-aggregate in typical multilamellar Lα lyotropic liquid-crystal nanostructures with sizes in the range of 100-200 nm and low polydispersities, very suitably fitted to remain in the bloodstream and cross the cell membrane. Interestingly, these nanoaggregates were able to compact, protect (from the degrading effect of DNase I), and transfect two DNA plasmids (pEGFP-C3, encoding the green fluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with an efficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/- charge ratio was maintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic because the viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosystem an optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experiments.

Supramolecular Control over the Interparticle Distance in Gold Nanoparticle Arrays by Cyclodextrin Polyrotaxanes.

Amphiphilic nonionic ligands, synthesized with a fixed hydrophobic moiety formed by a thiolated alkyl chain and an aromatic ring, and with a hydrophilic tail composed of a variable number of oxyethylene units, were used to functionalize spherical gold nanoparticles (AuNPs) in water. Steady-state and time-resolved fluorescence measurements of the AuNPs in the presence of α-cyclodextrin (α-CD) revealed the formation of supramolecular complexes between the ligand and macrocycle at the surface of the nanocrystals. The addition of α-CD induced the formation of inclusion complexes with a high apparent binding constant that decreased with the increasing oxyethylene chain length. The formation of polyrotaxanes at the surface of AuNPs, in which many α-CDs are trapped as hosts on the long and linear ligands, was demonstrated by the formation of large and homogeneous arrays of self-assembled AuNPs with hexagonal close packing, where the interparticle distance increased with the length of the oxyethylene chain. The estimated number of α-CDs per polyrotaxane suggests a high rigidization of the ligand upon complexation, allowing for nearly perfect control of the interparticle distance in the arrays. This degree of supramolecular control was extended to arrays formed by AuNPs stabilized with polyethylene glycol and even to binary arrays. Electromagnetic simulations showed that the enhancement and distribution of the electric field can be finely controlled in these plasmonic arrays.

Plasmid-Templated Control of DNA-Cyclodextrin Nanoparticle Morphology through Molecular Vector Design for Effective Gene Delivery.

Engineering self-assembled superstructures through complexation of plasmid DNA (pDNA) and single-isomer nanometric size macromolecules (molecular nanoparticles) is a promising strategy for gene delivery. Notably, the functionality and overall architecture of the vector can be precisely molded at the atomic level by chemical tailoring, thereby enabling unprecedented opportunities for structure/self-assembling/pDNA delivery relationship studies. Beyond this notion, by judiciously preorganizing the functional elements in cyclodextrin (CD)-based molecular nanoparticles through covalent dimerization, here we demonstrate that the morphology of the resulting nanocomplexes (CDplexes) can be tuned, from spherical to ellipsoidal, rod-type, or worm-like nanoparticles, which makes it possible to gain understanding of their shape-dependent transfection properties. The experimental findings are in agreement with a shift from chelate to cross-linking interactions on going from primary-face- to secondary-face-linked CD dimers, the pDNA partner acting as an active payload and as a template. Most interestingly, the transfection efficiency in different cells was shown to be differently impacted by modifications of the CDplex morphology, which has led to the identification of an optimal prototype for tissue-selective DNA delivery to the spleen in vivo.

Transfection of plasmid DNA by nanocarriers containing a gemini cationic lipid with an aromatic spacer or its monomeric counterpart.

This study performed a biophysical characterization (electrochemistry, structure and morphology) and assessment of the biological activity and cell biocompatibility of GCL/DOPE-pDNA lipoplexes comprised of plasmid DNA and a mixed lipid formed by a DOPE zwitterionic lipid and a gemini cationic lipid N-N'-(1,3-phenylene bis (methylene)) bis (N,N-dimethyl-N-(1-dodecyl) ammonium dibromide (12PH12) containing an aromatic spacer or its monomeric counterpart surfactant, N-benzyl-N,N-dimethyl-N-(1-dodecyl) ammonium bromide (12PH). Electrochemical results reveal that i) the gemini cationic lipid (12PH12) and the plasmid pDNA yield effective charges less than their nominal charges (+2 and -2/bp, respectively) and that ii) both vectors (12PH12/DOPE and 12PH/DOPE) could compact pDNA and protect it from DNase I degradation. SAXS and cryo-TEM experiments indicate the presence of a lamellar lyotropic liquid crystal phase represented as alternating layers of mixed lipid and plasmid. Transfection efficiency (by FACS and luminometry) and cell viability assay in COS-7 cells, performed with two plasmid DNAs (pEGFP-C3 and pCMV-Luc VR1216), confirm the goodness of the proposed formulations (12PH12/DOPE and 12PH/DOPE) to transport genetic material, with efficiencies and biocompatibilities comparable to or better than those exhibited by the control Lipofectamine 2000*. In conclusion, although major attention has been paid to gemini cationic lipids in the literature, due to the large variety of modifications that their structures may support to improve the biological activity of the resulting lipoplexes, it is remarkable that the monomeric counterpart surfactant with an aromatic group analyzed in the present work also exhibits good biological activity. The in vitro results reported here indicate that the optimum formulations of the gene vectors studied in this work efficiently transfect plasmid DNA with very low toxicity levels and, thus, may be used in forthcoming in vivo experiments.

A biophysical study of gene nanocarriers formed by anionic/zwitterionic mixed lipids and pillar[5]arene polycationic macrocycles.

The use of divalent cations as mediators between anionic lipids (ALs) and nucleic acids has been explored for several years in gene therapy. However, a promising anionic lipid system which could surpass the outcomes of current cationic lipids (CLs) has not been found yet. One plausible reason for such poor efficiencies may be the impossibility of AL-DNA lipoplexes mediated by divalent cations to reach charge inversion, in contrast with the usual behavior of CL-DNA lipoplexes. In the present study, divalent bridge-cations have been replaced by a multivalent positively charged macrocycle in order to see whether charge reversal is reached and how this fact may improve transfection efficiency (TE). For that purpose, an extensive biophysical and biochemical study has been carried out on lipoplexes constituted by a mixture of: (i) an anionic lipid DOPG (sodium salt of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)); (ii) a zwitterionic lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), which acts as a neutral helper lipid at physiological pH 7.4; (iii) a plasmid DNA (pDNA); and (iv) a polycationic macrocycle, pillar[5]arene (P10+), with the role of bridging the electrostatic interaction between the anionic mixed lipids and the pDNA, also negatively charged. The studies have been done at several DOPG molar compositions (α) and pillar[5]arene concentrations. Electrochemical experiments (zeta potential and gel electrophoresis) have revealed that, interestingly, DOPG/DOPE-P10+-pDNA lipoplexes show a charge inversion. Both studies have indicated that, at [P10+] ≥ 15 µM, pDNA is efficiently compacted by DOPG/DOPE mixed lipids, using P10+ as a bridge between the negative charge of the AL and anionic pDNA. SAXS diffractograms have shown the presence of two lyotropic liquid crystal phases: an inverted hexagonal one (H) found at low composition (α = 0.2), and a lamellar one (Lα) at medium composition (α = 0.5). Cryo-TEM and AFM experiments have confirmed these structures. Transfection and cell viability experiments using COS-7 cells in the presence of serum have reported moderate-to-high transfection levels and good cell viability results. The whole ensemble of the biophysical and biochemical results of the DOPG/DOPE-P10+-pDNA lipoplex indicates that this system may open up a novel and very promising route in the anionic non-viral gene vectors field.