Temporal control over cellular targeting through hybridization of folate-targeted dendrimers and PEG-PLA nanoparticles

Polymeric nanoparticles (NPs) and dendrimers are two major classes of nanomaterials that have demonstrated great potential for targeted drug delivery. However, their targeting efficacy has not yet met clinical needs, largely because of a lack of control over their targeting kinetics, which often results in rapid clearance and off-target drug delivery. To address this issue, we have designed a novel hybrid NP (nanohybrid) platform that allows targeting kinetics to be effectively controlled through hybridization of targeted dendrimers with polymeric NPs. Folate (FA)-targeted generation 4 poly(amidoamine) dendrimers were encapsulated into poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PLA) NPs using a double emulsion method, forming nanohybrids with a uniform size (~100 nm in diameter) at high encapsulation efficiencies (69-85%). Targeted dendrimers encapsulated within the NPs selectively interacted with FA receptor (FR)-overexpressing KB cells upon release in a temporally controlled manner. The targeting kinetics of the nanohybrids were modulated using three different molecular weights (MW) of the PLA block (23, 30, and 45 kDa). The release rates of the dendrimers from the nanohybrids were inversely proportional to the MW of the PLA block, which dictated their binding and internalization kinetics with KB cells. Our results provide evidence that selective cellular interactions can be kinetically controlled by the nanohybrid design, which can potentially enhance targeting efficacy of nanocarriers.     

Carboxymethyl chitosan-poly(amidoamine) dendrimer core–shell nanoparticles for intracellular lysozyme delivery

Intracellular delivery of native, active proteins is challenging due to the fragility of most proteins. Herein, a novel polymer/protein polyion complex (PIC) nanoparticle with core–shell structure was prepared. Carboxymethyl chitosan-grafted-terminal carboxyl group-poly(amidoamine) (CM-chitosan-PAMAM) dendrimers were synthesized by amidation and saponification reactions. ¹H NMR was used to characterize CM-chitosan-PAMAM dendrimers. The TEM images and results of lysozyme loading efficiency indicated that CM-chitosan-PAMAM dendrimers could self-assemble into core–shell nanoparticles, and lysozyme was efficiently encapsulated inside the core of CM-chitosan-PAMAM dendrimer nanoparticles. Activity of lysozyme was completely inhibited by CM-chitosan-PAMAM Dendrimers at physiological pH, whereas it was released into the medium and exhibited a significant enzymatic activity in an acidic intracellular environment. Moreover, the CM-chitosan-PAMAM dendrimer nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 3.16 mg/ml. The results indicated that these CM-chitosan-PAMAM dendrimers have excellent properties as highly potent and non-toxic intracellular protein carriers, which would create opportunities for novel applications in protein delivery.     

Biomimetics: From Bioinformatics to Rational Design of Dendrimers as Gene Carriers

Biomimetics, or the use of principles of Nature for developing new materials, is a paradigm that could help Nanomedicine tremendously. One of the current challenges in Nanomedicine is the rational design of new efficient and safer gene carriers. Poly(amidoamine) (PAMAM) dendrimers are a well-known class of nanoparticles, extensively used as non-viral nucleic acid carriers, due to their positively charged end-groups. Yet, there are still several aspects that can be improved for their successful application in in vitro and in vivo systems, including their affinity for nucleic acids as well as lowering their cytotoxicity. In the search of new functional groups that could be used as new dendrimer-reactive groups, we followed a biomimetic approach to determine the amino acids with highest prevalence in protein-DNA interactions. Then we introduced them individually as terminal groups of dendrimers, generating a new class of nanoparticles. Molecular dynamics studies of two systems: PAMAM-Arg and PAMAM-Lys were also performed in order to describe the formation of complexes with DNA. Results confirmed that the introduction of amino acids as terminal groups in a dendrimer increases their affinity for DNA and the interactions in the complexes were characterized at atomic level. We end up by briefly discussing additional modifications that can be made to PAMAM dendrimers to turned them into promising new gene carriers.     

Low‐generation asymmetric dendrimers exhibit minimal toxicity and effectively complex DNA

Conventional dendrimers are spherical symmetrically branched polymers ending with active surface functional groups. Polyamidoamine (PAMAM) dendrimers have been widely studied as gene delivery vectors and have proven effective at delivering DNA to cells in vitro. However, higher‐generation (G4‐G8) PAMAM dendrimers exhibit toxicity due to their high cationic charge density and this has limited their application in vitro and in vivo. Another limitation arises when attempts are made to functionalize spherical dendrimers as targeting moieties cannot be site‐specifically attached. Therefore, we propose that lower‐generation asymmetric dendrimers, which are likely devoid of toxicity and to which site‐specific attachment of targeting ligands can be achieved, would be a viable alternative to currently available dendrimers. We synthesized and characterized a series of peptide‐based asymmetric dendrimers and compared their toxicity profile and ability to condense DNA to spherical PAMAM G1 dendrimers. We show that asymmetric dendrimers are minimally toxic and condense DNA into stable toroids which have been reported necessary for efficient cell transfection. This paves the way for these systems to be conjugated with targeting ligands for gene delivery in vitro and in vivo. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Predictive evaluation for the preparation of a synthetic Y-shaped DNA nanostructure

With the advent of deoxyribonucleic acid (DNA) nanotechnology, the Y-shaped DNA nanostructure (Y-DNA) as a basic block was first created. Due to their characteristic selectivity and specificity, Y-DNA-based materials have been utilized in a variety of scientific fields including multiplexed nanobarcoding. Basically, the tripod DNA nanostructure was prepared by simple hybridization of three different single stranded DNA (ssDNA). Before the synthetic process, the optical densities (OD) of the three ssDNAs were measured to accurately estimate the concentration. Through repeated temperature fluctuations, three ssDNAs were hybridized into a Y-shaped block with both a central junction and three blunt ended arms. After the reaction, the ODs of the synthesized DNA products were measured and compared with the theoretical OD values calculated by a MATLAB program (‘matrix laboratory’) with different molar concentrations and volumes to predict the presence of Y-DNA. Simultaneously, the product was analyzed by agarose gel electrophoresis to confirm the YDNA structure. The measured ODs of the solutions with confirmed Y-DNA structures were close to the theoretical maximum OD values. This article provides means to help understand and prepare Y-DNA by performing OD measurements. It is highly expected that this guide will be an excellent starting point for structural DNA nanotechnology.     

Optical detection of DNA hybridization based on fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles

A novel system for the detection of DNA hybridization in a homogeneous format is developed. This method is based on fluorescence quenching by gold nanoparticles used as both nanoscaffolds for the immobilization of capture sequences and nanoquenchers of fluorophores attached to detection sequences. The oligonucleotide-functionalized gold nanoparticles are synthesized by derivatizing the colloidal gold solution with 5'-thiolated 12-base oligonucleotides. Introduction of sequence-specific target DNAs (24 bases) into the mixture containing dye-tagged detection sequences and oligonucleotide-functionalized gold nanoparticles results in the quenching of carboxytetramethylrhodamine-labeled DNA fluorescence because DNA hybridization occurs and brings fluorophores into close proximity with oligonucleotide-functionalized gold nanoparticles. The quenching efficiency of fluorescence increases with the target DNA concentration and provides a quantitative measurement of sequence-specific DNA in sample. A linearity is obtained within the range from 1.4 to 92 nM. The target sequence is detected down to 2 nM. This new system not only overcomes many of the drawbacks inherent in radioisotopic measurement or enzyme-linked assay but also avoids the requirement for the stem-loop structure compared with conventional molecular beacons. Furthermore, the background signal that is defined as fluorescence quenching arising from electrostatic attraction between positively charged fluorophores and negatively charged gold nanoparticles is comparatively low due to electrostatic repulsion between negatively charged oligonucleotides. In addition, this is a homogeneous assay that can offer the potential to be monitored in real time, be amenable to automation, eliminate washing steps, and reduce the risk of contamination.     

Characterization of a (Y;4) translocation by DNA hybridization

Summary A phenotypically normal male with azoospermia was found to have a translocation between the short arm of the Y chromosome and the distal long arm of a chromosome 4. By cytogenetic analysis it could not be determined whether the translocation was reciprocal, nor whether it was balanced. In situ DNA hybridization with two pseudoautosomal and one Y-specific probe demonstrated that the breakpoint was on distal Yp and that there was Y chromosome material on 4q. Thus the translocation was reciprocal and could be characterized as t(Y;4)(pll;q32). There was no evidence for loss of Y-DNA sequences as judged by Southern blotting with Y-DNA probes. Thus the translocation may be balanced. We conclude that DNA hybridization can be used to refine considerably the cytogenetic analysis of such translocations.     

Femtomolar DNA detection by parallel colorimetric darkfield microscopy of functionalized gold nanoparticles

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0×10(-14) M without the need for a spectrometer or a scanning stage.     

Assembly of myoglobin layer-by-layer films with poly(propyleneimine) dendrimer-stabilized gold nanoparticles and its application in electrochemical biosensing

The small-sized Au nanoparticles (3 nm) were prepared by reduction of HAuCl(4) in the presence of poly(propyleneimine) (PPI) dendrimers, forming the stable PPI-Au nanoclusters in aqueous medium. The PPI-Au nanoclusters might take a kind of "core-shell" structure, in which several PPI molecules were attached on the surface of one gold nanoparticle. The PPI-Au nanoclusters in aqueous dispersions and myoglobin (Mb) in its buffers at pH 5.0 were then alternately adsorbed on the surface of pyrolytic graphite (PG) electrodes and other solid substrates, forming {PPI-Au/Mb}(n) layer-by-layer films, which was confirmed by cyclic voltammetry (CV) and quartz crystal microbalance (QCM). {PPI-Au/Mb}(n) films on PG electrodes demonstrated a pair of well-defined and quasi-reversible CV reduction-oxidation peaks for Mb heme Fe(III)/Fe(II) couple and good electrocatalytic properties toward reduction of oxygen and hydrogen peroxide. Compared with {Au/Mb}(n) multilayer films containing no dendrimers and {PAMAM/Mb}(n) films assembled by polyamidoamine (PAMAM) dendrimers and Mb but in the absence of Au nanoparticles, {PPI-Au/Mb}(n) films showed better electrochemical behaviors and catalytic performances, which may be attributed to the unique structure of PPI-Au nanoclusters and good conductivity of gold nanoparticles. This novel kind of protein multilayer films assembled with dendrimer-stabilized gold nanoparticles may provide a new and general approach to fabricate the biosensors and bioreactors based on the direct electrochemistry of proteins or enzymes.     

Chitosan-reduced gold nanoparticles: a novel carrier for the preparation of spray-dried liposomes for topical delivery

Exposure of skin to various chemical and physical agents results in excessive stress to the outermost cell layer of the skin, causing different degenerative effects that can be minimized by using antioxidant formulations. The major challenge, in this regard, is to develop a formulation, which can prevent photodegradation of the actives, thus allowing a significant amount to be deposited at the site. In recent decades, liposomal formulations have been extensively employed to overcome the barrier properties of the skin and photodegradation of actives. In the present study, chitosan-reduced gold nanoparticles were investigated for its potential as a carrier to prepare liposomes by a spray-drying method. Liposomes so obtained were characterized for phospholipid recovery, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, particle size, zeta potential, encapsulation efficiency, and deposition of drug and gold nanoparticles in the rat skin. Further, a liposomal gel formulation was prepared using Carbopol? 980 NF (Noveon Systems, Kochi, India) and evaluated for drug deposition in the skin. Antioxidant activity of vitamin C encapsulated in gold liposomes was determined on a human leukemia (HL-60) cell line. The use of gold nanoparticles as a carrier showed improved phospholipid recovery and thus overcomes the liposome scalability problem. DRIFT spectra confirmed the presence of phospholipid in the formulation. Liposomal gel showed improved drug deposition, as compared to control and marketed preparations. A more interesting contribution of the chitosan-reduced gold nanoparticles was an enhanced antioxidant activity seen in case of the vitamin C-loaded gold liposomal formulation. Liposomal formulation was found to be stable for 3 months at 30°C and 65% relative humidity.     

STUDIES ON THE ATTACHMENT OF DNA TO SILICA-COATED NANOPARTICLES THROUGH A DIELS-ALDER REACTION

A new method has been investigated for the functionalization of gold nanoparticles with DNA. Silica-coated nanoparticles functionalized with a maleimide have been prepared. These particles are designed to react with modified DNA containing a diene functionality at one end of the molecule. The result would be the formation of a more stable attachment of the DNA to the particle through a Diels-Alder reaction. This covalent attachment would not be susceptible to ligand exchanges, which are known to occur in the conventional DNA functionalization of gold nanoparticles.     

Studies on the attachment of DNA to silica-coated nanoparticles through a Diels-Alder reaction

A new method has been investigated for the functionalization of gold nanoparticles with DNA. Silica-coated nanoparticles functionalized with a maleimide have been prepared. These particles are designed to react with modified DNA containing a diene functionality at one end of the molecule. The result would be the formation of a more stable attachment of the DNA to the particle through a Diels-Alder reaction. This covalent attachment would not be susceptible to ligand exchanges, which are known to occur in the conventional DNA functionalization of gold nanoparticles.     

Structurally flexible triethanolamine core PAMAM dendrimers are effective nanovectors for DNA transfection in vitro and in vivo to the mouse thymus

With the aim of developing dendrimer nanovectors with a precisely controlled architecture and flexible structure for DNA transfection, we designed PAMAM dendrimers bearing a triethanolamine (TEA) core, with branching units pointing away from the center to create void spaces, reduce steric congestion, and increase water accessibility for the benefit of DNA delivery. These dendrimers are shown to form stable nanoparticles with DNA, promote cell uptake mainly via macropinocytosis, and act as effective nanovectors for DNA transfection in vitro on epithelial and fibroblast cells and, most importantly, in vivo in the mouse thymus, an exceedingly challenging organ for immune gene therapy. Collectively, these results validate our rational design approach of structurally flexible dendrimers with a chemically defined structure as effective nanovectors for gene delivery, and demonstrate the potential of these dendrimers in intrathymus gene delivery for future applications in immune gene therapy.     

Exonuclease III-based and gold nanoparticle-assisted DNA detection with dual signal amplification

Herein we report a sensitive electrochemical biosensor for DNA detection by making use of exonuclease III and probe DNA functionalized gold nanoparticles. While probe DNA P1 modified on a gold electrode surface can self-hybridize into a stem-loop structure with an exonuclease III-resistant 3' overhang end, in the presence of target DNA, P1 may also hybridize with the target DNA to form a duplex region. Therefore, exonuclease III may selectively digest P1 from its 3'-hydroxyl termini until the duplex is fully consumed. Since a single target DNA can trigger exonuclease III digestion of numerous P1 strands, the first signal amplification is achieved. On the other hand, since the digested P1, exposing its complementary sequence to probe DNA P2, can further hybridize with P2 that has been previously modified on the surface of gold nanoparticles, many nanoparticles loaded with numerous DNA strands are immobilized onto the electrode surface. Consequently, large amount of electroactive molecules [Ru(NH(3))(6)](3+) can bind with the DNA strands to produce an intense electrochemical response as the second signal amplification. Based on the studies with cyclic voltammetry (CV) and chronocoulometry (CC) techniques, the proposed biosensor can sensitively detect specific target DNA at a picomolar level with high specificity.     

Hemocompatible poly(NIPAm-MBA-AMPS) colloidal nanoparticles as carriers of anti-inflammatory cell penetrating peptides

Anionic copolymer systems containing sulfated monomers have great potential for delivery of cationic therapeutics, but N-isopropylacrylamide (NIPAm) 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) copolymer nanoparticles have seen limited characterization to date with regard to physical properties relevant to loading and release of therapeutics. Characterization of polymeric nanoparticles incorporating AMPS showed an increased size and decreased thermodynamic swelling ratios of AMPS containing particles as compared to NIPAm nanoparticles lacking AMPS. Particles with increasing AMPS addition showed an increased propensity for uniformity, intraparticle colloidal stability, and drug loading capacity. Peptide encapsulated in particles was shielded from peptide degradation in serum. Particles were shown not impede blood coagulation or to cause hemolysis. This study has demonstrated that AMPS incorporation into traditional NIPAm nanoparticles presents a tunable parameter for changing particle LCST, size, swelling ratio, ζ potential, and cationic peptide loading potential. This one-pot synthesis results in a thermosensitive anionic nanoparticle system that is a potentially useful platform to deliver cationic cell penetrating peptides.     

Colloidal systems for drug delivery: from design to therapy

Nanomedicine, or medicine using nanometric devices, has emerged in the past decade as an exhilarating domain that can help to solve a number of problems linked to unsatisfactory therapeutic responses of so-called 'old drugs'. This dissatisfaction stems from inadequate biodistribution after a drug's application, which leads to a limited therapeutic response but also to numerous side effects to healthy organs. The biodistribution of drugs encapsulated in a nanoobject that will act as a vector can be modified to tune its therapeutic efficacy. This review provides a general overview of existing colloidal nanovectors: liposomes, polymeric micelles, polymeric vesicles, polymeric nanoparticles (NPs), and dendrimers. We describe their characteristics, advantages and drawbacks, and discuss their use in the treatment of various diseases.     

纳米金增强复杂体系中PCR扩增低拷贝基因的反应特异性初步研究

目的：利用纳米金颗粒提高复杂体系基因组低拷贝基因PCR扩增的反应特异性。方法：首先,模拟复杂基因组扩增模式体系,以接近单拷贝的λDNA为模板,在PCR过程中加入纳米金颗粒,设计优化实验,以便模拟建立复杂基因组低拷贝目的基因PCR扩增的模式体系。随后,扩增人类基因组的疾病相关的低拷贝基因模板（如人基因组肿瘤坏死因子基因外显子1的380bp）,以检验纳米金优化增强PCR反应特异性的实际效果。结果：在复杂体系基因组的低拷贝基因PCR扩增中,纳米金颗粒能够较好地增强其PCR反应的特异性。结论：初步表明基于纳米金的纳米粒子PCR方法可以对复杂的实际基因组体系低拷贝基因的PCR扩增起到优化作用,这对于PCR反应优化方法的改进、推广具有重要的参考价值。     

Assays Based on Differential Adsorption of Single-stranded and Double-stranded DNA on Unfunctionalized Gold Nanoparticles in a Colloidal Suspension

Under certain conditions, single-stranded DNA adsorbs to negatively charged gold nanoparticles in a colloid whereas double-stranded DNA does not. We present evidence that this phenomenon can be explained by the difference in their electrostatic properties that in turn reflects conformational differences. The ability to discriminate the hybridization state of DNA on the basis of adsorption behavior can be utilized to design simple colorimetric and fluorimetric assays that take advantage of plasmon resonance in the gold nanoparticles. We present examples where we detect specific target sequences in oligonucleotides and in genomic DNA. Because conformational changes in special DNA sequences can also be induced by analytes such as potassium, we report a potassium ion detection scheme based on the same principle.     

Efficient orthogonal bioconjugation of dendrimers for synthesis of bioactive nanoparticles

Nanoparticles carrying biologically active functional sets (e.g., targeting moiety, payload, tracer) have potential use in a wide range of clinical applications. Though complex, such constructions should, as far as possible, have a defined molecular architecture and be monodisperse. However, the existing methods to achieve this goal are unsuitable for the incorporation of peptides and proteins, and those that provide for orthogonal introduction of two different types of functional element are incompatible with the use of commercially available materials. In this study, we have developed approaches for the production of nanoparticles based on commercially available polyamidoamine (PAMAM) dendrimers. First, we identified an optimized oxime conjugation strategy under which complex dendrimers can be fully decorated not only with model peptides, but also with recombinant proteins (insulin was taken as an example). Second, we developed a strategy based on a two-chain covalent heterodendrimer (a "diblock") based on cystamine core PAMAM dendrimers and used it to generate heterodendrimers, into which a peptide array and a mannose array were orthogonally introduced. Finally, by incorporating a functionalized linker into the diblock architecture we were able to site-specifically introduce a third functional element into the nanoparticle. We exemplified this approach using fluorescein, a mannose array, and a peptide array as the three functionalities. We showed that incorporation of a mannose array into a nanoparticle strongly and specifically enhances uptake by sentinel cells of the immune system, an important property for vaccine delivery applications. These PAMAM dendrimer-based approaches represent a robust and versatile platform for the development of bioactive nanoparticles.     

Chitosan-reduced gold nanoparticles: a novel carrier for the preparation of spray-dried liposomes for topical delivery

Exposure of skin to various chemical and physical agents results in excessive stress to the outermost cell layer of the skin, causing different degenerative effects that can be minimized by using antioxidant formulations. The major challenge, in this regard, is to develop a formulation, which can prevent photodegradation of the actives, thus allowing a significant amount to be deposited at the site. In recent decades, liposomal formulations have been extensively employed to overcome the barrier properties of the skin and photodegradation of actives. In the present study, chitosan-reduced gold nanoparticles were investigated for its potential as a carrier to prepare liposomes by a spray-drying method. Liposomes so obtained were characterized for phospholipid recovery, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, particle size, zeta potential, encapsulation efficiency, and deposition of drug and gold nanoparticles in the rat skin. Further, a liposomal gel formulation was prepared using Carbopol^® 980 NF (Noveon Systems, Kochi, India) and evaluated for drug deposition in the skin. Antioxidant activity of vitamin C encapsulated in gold liposomes was determined on a human leukemia (HL-60) cell line. The use of gold nanoparticles as a carrier showed improved phospholipid recovery and thus overcomes the liposome scalability problem. DRIFT spectra confirmed the presence of phospholipid in the formulation. Liposomal gel showed improved drug deposition, as compared to control and marketed preparations. A more interesting contribution of the chitosan-reduced gold nanoparticles was an enhanced antioxidant activity seen in case of the vitamin C–loaded gold liposomal formulation. Liposomal formulation was found to be stable for 3 months at 30°C and 65% relative humidity.