Strategies for ocular siRNA delivery: Potential and limitations of non-viral nanocarriers
© Thakur et al.; licensee BioMed Central Ltd. 2012
Received: 11 November 2011
Accepted: 26 April 2012
Published: 1 December 2012
Controlling gene expression via small interfering RNA (siRNA) has opened the doors to a plethora of therapeutic possibilities, with many currently in the pipelines of drug development for various ocular diseases. Despite the potential of siRNA technologies, barriers to intracellular delivery significantly limit their clinical efficacy. However, recent progress in the field of drug delivery strongly suggests that targeted manipulation of gene expression via siRNA delivered through nanocarriers can have an enormous impact on improving therapeutic outcomes for ophthalmic applications. Particularly, synthetic nanocarriers have demonstrated their suitability as a customizable multifunctional platform for the targeted intracellular delivery of siRNA and other hydrophilic and hydrophobic drugs in ocular applications. We predict that synthetic nanocarriers will simultaneously increase drug bioavailability, while reducing side effects and the need for repeated intraocular injections. This review will discuss the recent advances in ocular siRNA delivery via non-viral nanocarriers and the potential and limitations of various strategies for the development of a ‘universal’ siRNA delivery system for clinical applications.
KeywordsBiomaterials siRNA Drug delivery Endosomal escape Nanocarriers Ocular siRNA delivery RNAi
Challenges of posterior segment ophthalmic therapeutics
Local and systemic routes for drug delivery
It is estimated that following instillation, only 5% of topically applied drugs enter the anterior chamber of the eye, either through trans-corneal permeation (Figure1, arrow 1) or non-corneal permeation into the anterior uvea through the conjunctiva and sclera (Figure1, arrow 2). Increasing the residence time on the eye through viscous formulation can slightly improve uptake. However, due to the physical barrier created by the corneal and conjunctival epithelium, and the relatively small tear volume (~7 μl) available, a maximal attainable absorption into the anterior chamber appears to be approximately 10% of the applied dose. Drugs are eliminated from the aqueous humor via aqueous turnover through the Schlemm’s canal and trabecular meshwork (Figure1, arrow 4) and by uptake into systemic circulation through uveoscleral blood flow (Figure1, arrow 5). Elimination via the first route occurs through convective flow at a rate of approximately 3 μl/min and is independent of drug type. Clearance through uveal blood flow however, is influenced by the ability of the drug to penetrate the endothelial walls of the blood vessels. Thus, lipophilic drugs clear more rapidly than hydrophilic drugs, often in the range of 20 – 30 μl/min. Coupled with the physical barrier created by the lens, flow of drugs from the anterior chamber to the posterior segment of the eye is negligible. Therefore, topical drug administration is typically limited to anterior complications. The systemic route is also severely limited in its ability to effectively deliver drugs to the back of the eye. Only an estimated 1 – 2% of compounds delivered via this route successfully cross the BAB (Figure1, arrow 3) and the BRB (Figure1, arrow 6) and accumulate within the retinal tissues. With many newly developed pharmaceuticals being protein-based, oral formulations become increasingly difficult to administer, as the drugs need to be protected from degradation within the gastro-intestinal tract. Furthermore, the large concentrations of drug required to achieve therapeutically relevant concentrations within the retinal tissues and the increased potential for off-target interactions makes oral administration an undesirable route of delivery for posterior segment therapies.
Intravitreal drug delivery
The most efficient means to deliver drugs into the posterior segment is through direct injection into the vitreous cavity (Figure1, arrow 7). Using a high-gauge needle, therapeutics may be introduced into the vitreous through simple injection, producing high concentrations of drug locally surrounding the retinal tissues while limiting off-target exposure. However, the concentration of drug is rapidly depleted from the posterior segment via permeation across the BRB (Figure1, arrow 8) and by diffusion across the vitreous to the anterior chamber (Figure1, arrow 9), which allows drugs to be cleared through the anterior route. Thus, repeat injections are required, often every 4 – 6 weeks, to maintain therapeutic concentrations of drug within the posterior segment. Repeat instillations are associated with increasing risk of injection-related complications, such as raised intraocular pressure, vitreous or retinal hemorrhage, retinal detachment, retinal tears, endophthalmitis, cataracts, floaters and transient blurry vision. Rates of endophthalmitis and cataract formation per injection are 0.2% and 0.05% respectively. Repeat injections are also associated with patient discomfort and adherence issues. Therefore, while intravitreal injections have the greatest clinical efficacy, they are also the most risky.
Currently, the most promising solutions to combat the challenges of posterior segment drug delivery are approaches that successfully utilize direct intravitreal delivery and sustain therapeutic concentrations for extended periods of time, thereby decreasing the frequency of intervention. The first commercially successful sustained release intravitreal device for treatment of cytomegalovirus retinitis was Vitrasert (Bausch and Lomb), a non-degrading implant that is surgically implanted at the pars plana. Vitrasert is a US Food and Drug Administration (FDA) approved drug delivery system, which consists of a tablet of ganciclovir coated with polyvinyl alcohol (PVA) and ethylene vinyl acetate (EVA). The impermeable EVA coating limits the surface area through which ganciclovir can release, forcing drug to diffuse through the small PVA rate-limiting membrane, slowing the release and allowing treatment for a period of 5 to 8 months. However, Vitrasert is a relatively large non-degrading device and therefore requires an incision for introduction into the vitreous cavity, as well as a secondary surgical intervention for device removal following exhaustion of the drug reservoir. The I-vation (Surmodics) drug delivery system is another example of a non-degrading, sustained intravitreal release device for the treatment of diabetic macular edema. The helical construct was designed to facilitate ease of implantation and removal, maximize surface area available for drug release, and allow sutureless anchorage within the vitreous. The titanium helix is coated with a blend of poly(methyl methacrylate) and EVA, which is loaded with triamcinolone acetonide and provides sustained release for 18–36 months[5, 8]. In contrast, the Iluvien (Alimera Sciences) drug delivery system consists of a very small cylindrical polyimide rod loaded with fluocinolone acetonide (FAc) capable of being injected through a 25-gauge needle and releasing low levels of drug for up to 3 years[5, 8]. However, as this scaffold is composed of non-degrading materials and is not fixed to the eye wall, it is expected to remain within the patient’s orbit following depletion of the drug and is currently under review by the FDA. Ozurdex (Allergan), an FDA approved dexamethasone loaded intravitreal insert for the treatment of macular edema and noninfectious uveitis, is another scaffold capable of introduction into the vitreous via minimally invasive injection using a 22-gauge applicator. However, unlike Iluvien, Ozurdex is composed of degradable poly(lactide-co-glycolide), thereby allowing scaffold degradation and clearance from the eye and body without the need for secondary surgical intervention.
With recent advances in pharmaceuticals, including regulatory approval of multiple pharmacotherapies to treat wet age-related macular degeneration (AMD), and the increasingly elderly demographic at risk of degenerative eye disorders, there has been renewed interest in designing novel drug delivery platforms, particularly nanocarriers, to address the limitations of posterior segment therapeutics. Furthermore, scientific research is continuing to shed new light on the fundamental biochemical pathways implicated in retinal degenerative diseases, which is leading to the discovery of new pharmacological targets and the development of novel therapeutics.
RNA interference and siRNA delivery
RNA interference (RNAi) is an evolutionarily conserved mechanism that has been observed in most organisms from plants to vertebrates. It is a mechanism that leads to sequence-specific post-transcriptional gene silencing that was first documented in animals by Andrew Fire and Craig Mello in 1998, both of whom subsequently received the Nobel Prize in Physiology or Medicine in 2006[12, 13].
RNA interference can provide a novel therapeutic modality to treat many human diseases by interfering with disease-causing and disease-promoting genes in a sequence-specific manner. Elbashir et al. were the first to demonstrate that small interfering RNA (siRNAs) can induce the RNAi pathway in mammalian cells without producing an adverse immune response. This immediately suggested that the RNAi pathway could potentially be manipulated in humans for the treatment of many human diseases. Theoretically, RNAi can be used to selectively alter the expression of any transcribed gene. This new paradigm in therapeutics allows one to address disease states previously considered ‘undruggable’. In addition, it creates new opportunities to alter important cellular processes such as cell division and apoptosis, both of which are significantly altered in many cancers.
RNA interference is essentially a conserved cellular mechanism that leads to post-transcriptional gene silencing, which can be manipulated for therapeutic applications in humans. Post-transcriptional gene silencing strategies can be broadly divided into four types: 1) single-stranded antisense oligodeoxynucleotides (ODNs)- synthetic molecules that can specifically hybridize with complementary mRNA and sterically inhibit protein translation, 2) ribozymes- catalytically active small RNA molecules that can specifically recognize and cleave single-stranded regions in RNA, 3) microRNA (miRNA)- endogenous, short double-stranded non-coding RNA molecules that play an important role in health and disease by modulating gene expression, and 4) siRNAs- these 18–25 nucleotide long duplexes are potent activators of the innate immune system that have been shown to initiate sequence-specific post-transcriptional gene silencing. Although all of these strategies can potentially be applied to suppress mRNA translation, it is generally accepted that siRNA technology offers the best combination of specificity, potency and versatility as a therapeutic. In addition, siRNAs are easily synthesized and do not require cellular expression systems or complex protein purification systems, making this technology significantly more cost effective over other small molecule therapeutics.
Small-interfering RNA mediates its post-transcriptional gene silencing effects via the RNAi pathway. In brief, when exogenous siRNA duplexes are introduced into mammalian cells, the 5’-end is phosphorylated. This duplex is then assembled into a multiprotein complex called RNA-induced silencing complex (RISC), which includes proteins such as Argonaute 2 (AGO2), Dicer, TRBP (HIV-1 TAR RNA-binding protein) and PACT (dsRNA-binding protein). The sense strand is then cleaved and unwound, leaving only the antisense strand associated with AGO2. Argonaute 2 is an endonuclease that promotes hybridization of this antisense strand to complementary cellular mRNAs and subsequent cleavage of the mRNA target. This results in ‘knocking down’ the translation of the target gene.
In designing siRNAs, the three most important attributes to be taken into account are: potency (effectiveness of gene silencing at low siRNA concentrations), specificity (minimize homology to other mRNAs) and nuclease stability (resistance against exonuclease and endonuclease activity). Moreover, there are two types of off-target effects that should be minimized: immune stimulation arising from siRNA recognition by the innate immune system, and unintended silencing of genes that share partial homology with the siRNA[15, 17].
It is clear that siRNA technology has a great therapeutic potential in medicine. However, one of the major limitations for their application in vitro and in vivo is the inability of siRNA to cross cell membranes and reach the cytoplasm. The negative charges arising from the phosphate groups in the siRNA backbone electrostatically repel negatively charged cell membranes, therefore limiting siRNA ability to diffuse across cell membranes. In addition, other challenges common to most drug delivery systems, including high molecular weight, short blood half-life, poor specificity and uptake in target tissues, cellular toxicity, and undesirable off-target effects, significantly hamper the successful application of siRNA therapeutics in medicine. Moreover, the intrinsic physical barriers, efficient drug clearance mechanisms and other complexities of ocular tissues such as the retina and the cornea pose a significant challenge to ocular siRNA delivery. In order to address these problems, several siRNA delivery strategies have been developed for in vitro and in vivo applications.
Numerous non-viral carriers including natural and synthetic polymers, polyplexes, liposomes, lipoplexes, peptides, dendrimers and free nucleic acid pressurized hydrodynamic injections, as well as virus-based vectors and plasmids encoding for siRNA, have been proposed for siRNA delivery. Although most of these strategies have been attempted with various degrees of success in vitro and in vivo, strategies for targeted siRNA delivery that are most relevant to ophthalmic applications will be reviewed.
Non-viral siRNA delivery systems
Although many types of polymers have been used to deliver oligonucleotides, much attention has focused on using cationic polymers for two main reasons: 1) their ability to electrostatically bind siRNA without the need for covalent attachment or encapsulation, and 2) the ability of amine containing cationic polymers to provide endosomal buffering and escape for intracytosolic siRNA delivery. Polyethylenimine (PEI) is perhaps the most investigated synthetic cationic polymer for nucleic acid delivery due to its uniquely high buffering capability at endosomal pH, known as the ‘proton sponge’ effect, which releases nucleic acid payloads into the cytoplasm after endocytosis. Grayson et al. have demonstrated that polyplexes of PEI can effectively deliver siRNA to cells in vitro. Kim et al. were among the first to employ the use of pegylated (PEG) PEI-siRNA cationic polyplexes targeted against vascular endothelial growth factor-A (VEGFA), vascular endothelial growth factor receptor-1 (VEGFR1) and/or VEGFR2 to significantly reduce herpes simplex virus-induced angiogenesis and stromal keratitis in murine ocular tissues in vivo. Notably, these PEG-PEI-siRNA polyplexes were effective in both local and systemic administration of the formulation. Given that PEI-siRNA has been successfully tested in vivo for the treatment of various diseases, it is a promising candidate as a nanocarrier for ocular siRNA delivery.
Dendrimers represent a group of nanoscale materials that are hyperbranched, monodisperse and have defined molecular weights. Structurally, dendrimers are composed of a central core, repeating units that make up the branches, and surface functional groups. Dendrimers are synthesized in a step-by-step fashion by the sequential addition of repeating units organized in concentric layers, called generations, around the central core. High generation dendrimers have numerous cavities within their hyperbranched structure to allow for the encapsulation of therapeutic agents such as siRNA molecules. The most common dendrimers used for siRNA delivery include poly(amidoamine) (PAMAM) and poly(propylene imine) (PPI). However, other types of dendrimers composed of amine-containing cationic polymers such as poly-L-lysine have been investigated for ODN (anti-VEGF) delivery to RPE cells in vitro, and have demonstrated long-term (4–6 months) inhibition (up to 95%) of laser-induced CNV after intravitreal injection in a rat model, without any observable adverse effects. The major advantages of dendrimers include biodegradability, ease of synthesis and customizability, such that they can be synthesized in various sizes and differing number and type of surface functional groups to optimize siRNA delivery. Recently, Agrawal et al. have developed dendrimer-conjugated magnetofluorescent nanoworms called ‘dendriworms’ that significantly enhance intracellular siRNA delivery in a mouse model by optimizing endosomal escape. Alternatively, Han et al. have conjugated CPPs, such as HIV transactivator of transcription (TaT), to PAMAM dendrimers for enhanced intracellular siRNA delivery in vitro and in vivo. Together, these results suggest that dendrimers are ideally suited to serve as nanocarriers, which can be loaded with siRNA and functionalized with PEG and targeting ligands for clinical applications. However, at present, there are no examples of dendrimeric siRNA delivery for ocular applications in the literature.
Clinical trials involving siRNA therapeutics for ocular diseases
Silence Therapeutics/ Quark/Pfizer
PF-655 (formerly REDD14NP and RTP801i)
RTP801/ DNA-damage- inducible transcript 4 gene (DDIT4)
Phase II – completed
Silence Therapeutics/ Quark/Pfizer
PF-655 (formerly REDD14NP and RTP801i)
RTP801/DNA- damage-inducible transcript 4 gene (DDIT4)
Phase II – terminated
Phase II- terminated
Glaucoma, Ocular hypertension
Non-arteritic ischemic optic neuropathy (NAION), Chronic optic nerve atropy, Glaucoma
Phase I – on going
(Quark Pharmaceuticals 2011b)
Chemically modified siRNAs
Various molecular locations on siRNA molecules can be chemically altered to resist hydrolysis and enhance cellular uptake. In order to increase the efficacy of siRNA delivery, much research has focused on increasing the nuclease resistance and therefore serum stability of siRNAs. Nucleases such as eri-1 are involved in the degradation of unmodified siRNA duplexes, which have been reported to have a short serum half-life of about 3–5 min. However, it has been shown that siRNA serum half-life can be extended up to 72 h with fully modified duplexes.
Among the multitude of possible siRNA modifications, there are two schools of thought regarding the best approach to developing chemically modified siRNA. In one approach, it is believed that extensive chemical modification of siRNA is most likely to lead to the greatest efficacy. For example, Sirna Therapeutics has several patents and products that favour extensive siRNA duplex modifications, where the sense and antisense strands have modified bases (2’-Fluoro-RNA pyrimidines (2’-F-RNA), DNA purines), altered covalent links between the nucleotides (phosphorothioate linkage (PS)) and inverted 5’ and 3’ abasic end caps. These extensive siRNA modifications translated into increased potency and a much longer serum half-life (48–72 h) in a Hepatitis B virus mouse model. In contrast, the other school of thought is focused on creating stabilized siRNAs with minimal modifications. For example, Alnylam Pharmaceuticals has many siRNA products that are selectively modified (2’-sugar modifications such as 2’-O-methyl or 2’-F-RNA) at vulnerable sites, such as those susceptible to endonuclease cleavage[15, 17, 57]. It is important to note that modifications to the RNA backbone can potentially impair siRNA-induced silencing activity, thus many reported modifications have been limited to the sense strand[58, 59]. However, the rules for predicting siRNA stability and potency are still unclear since some studies have demonstrated antisense modifications with preserved siRNA functionality[60, 61], while other studies have shown sense strand modifications with reduced siRNA efficiency[62, 63].
Various chemical modifications to the terminals, backbone, nucleobases and sugars of siRNAs can be implemented to protect the duplex from exonuclease degradation. For example, the phosphodiester (PO4) linkages along the RNA backbone can be replaced with PS or boranophosphonate (PB) at the 3’ end[64–66]. It has been shown that PS derived oligonucleotides stimulate the physical uptake of siRNA in human cells, while siRNAs with PB backbone modifications have less cytotoxicity and a much higher nuclease resistance than native siRNA. Such PB siRNAs are at least 10 times more nuclease resistant than unmodified siRNAs, and have recently been used to treat patients with AMD. The process has reached Phase II clinical trials, and it was found to have no observable side effects. Replacement of sugar moieties at the 2’-hydroxyl group of the ribose backbone with 2’-O-methyl, 2’-fluoro, or 2’-methoxyethyl groups can further improve in vivo stability[66, 69]. Moreover, various molecules can be conjugated to the 5’ or 3’ ends of the sense strand, without affecting the activity of the antisense strand needed for silencing. This method can allow for cell specific targeting or visualization of siRNA uptake and distribution by introducing appropriate ligands and fluorophores respectively. However, degradation of these artificially altered siRNA molecules may result in metabolites with unsafe or otherwise unwanted reactivity. Chemical modification of siRNA can increase stability in biological solutions, target specificity and potency. However, the benefits of modification must be measured against the cost and labour of the modification process, as well as its effects on immune stimulation, which are generally difficult to predict and require empirical testing in vivo.
Immune stimulation and other off-target effects of siRNA delivery
In addition to the gene knockdown effects of the RNAi pathway, there are many other potential consequences that can be initiated by siRNA in vivo. Hence, these so called ‘off-target effects’ need to be considered and evaluated in any siRNA delivery study. For example, it is well known that double stranded RNA (dsRNAs) greater than 30 bp are potent activators of the innate immune response. Although siRNA duplexes are shorter than 30 bp, many recent studies have begun reporting off-target effects[58, 71]. In general, RNAs are recognized by three major types of immunoreceptors: Toll-like Receptors (TLR), protein kinase R (PKR) and helicases. Toll-like receptors are found on cell-surfaces (TLR3) and in endosomes (TLR3,7,8), whereas PKR and helicases (MDA5, RIG-I) are found in the cytoplasm[72, 73]. Immune recognition can lead to a host of downstream effects at the cellular level, including cytokine release, interferon response and changes in gene expression. At the whole body level, the use of unmodified siRNAs have been known to induce systemic toxicity, increase serum transaminases, decrease body weight, lymphopenia and piloerection. Thus, proper siRNA design should likely incorporate features to minimize the possibility of undesirable immune activation.
Although immune activation is influenced by many factors such as oligonucleotide length, sequence, chemical modification, mode of delivery and immune cell type involved, it has been previously shown that chemically modified siRNAs can be synthesized so as to reduce their immunostimulatory properties. However, it is interesting to note that immune stimulation may also have desirable consequences, such as anti-angiogenesis via the TLR3 pathway. Although this type of therapeutic immune stimulation may be useful from the standpoint of treating cancer, it can also have potentially severe side-effects.
In addition to immune stimulation, other off-target effects can originate from the partial hybridization of the antisense strand of siRNA with an unintended mRNA. This may lead to the cleavage and subsequent knockdown of the wrong gene. In addition, siRNAs can have their sense strand incorporated into RISC, leading to other off-target effects. To address these problems, siRNA sequences can be carefully selected to minimize complementarity with unwanted mRNAs, and chemically modified siRNAs can be used to increase the selective incorporation of the antisense strand into RISC[62, 76]. This highlights the importance of proper siRNA design in mediating target gene knockdown.
Cellular uptake of nanocarriers and endosomal escape strategies
After internalization of nanocarriers into cells, many studies have shown that large fractions of these nanocarriers can remain sequestered in trafficking vesicles and endolysosomes. This implies that some types of nanocarriers may not be suitable for delivering membrane-impermeable therapeutics (such as siRNA) to intracellular targets. Moreover, a lysosomal localization of unmodified naked siRNA will likely result in the degradation of siRNA. Hence, much research has focused on intracellular delivery strategies such as cationic lipid transfection, microinjection and electroporation. However, most of these strategies are limited to in vitro conditions due to their invasiveness, variable transfection efficiency, complexity of the procedure and potential for altering/disrupting cellular function. Recent efforts have demonstrated that endosomal escape strategies can be incorporated into nanocarrier design to significantly enhance cytosolic delivery of siRNA. Most commonly, CPPs, pH responsive polymers, fusogenic peptide sequences and hydrophobic molecules have been used for nanocarrier endosomal escape. Nanocarriers functionalized with CPPs such as the TaT, VP22, penetratin and polyarginine have been shown to permeate through the plasma membrane for direct cytoplasmic delivery[83–86]. Alternatively, other pH-responsive approaches tend to induce the ‘proton-sponge effect’ for endosomal escape via the clever use of cationic protonable amine-containing polymers such as PEI. In this approach, PEI acts as a buffer against endolysosomal acidification and causes the osmotic swelling and rupture of endolysosomes, releasing the nanocarriers into the cytosol (Figure4). In contrast, other approaches attempt to conjugate drugs to fusogenic peptide sequences, such as GALA and KALA, or hydrophobic molecules such that the nanocarrier can traverse membranes. For example, cholesterol-tagging has been shown to improve cytosolic delivery of siRNA with minimal cytotoxicity.
Interestingly, lipid-based nanocarriers can also be engineered to fuse with cell membranes, either avoiding endocytosis completely or escaping endolysosomes without inducing endolysosomal lysis. Although some studies suggest that a net positive surface charge and a high cationic lipid/siRNA molar charge ratio are important factors required to facilitate efficient membrane fusion with lipid-based nanocarriers, it has been reported that these factors also seem to significantly increase toxicity. Recently, Leal et al. have reported the development of cationic liposome (CL)-siRNA complexes with novel cubic phase nanostructures, which offer a novel solution to lipid based delivery. Cubic phase lipid delivery systems readily fuse with cell membranes due to their high charge density and positive Gaussian modulus, delivering their cargo through transiently induced pores in the endosomal membrane, which results in highly efficient gene silencing in vitro with low toxicity[91, 92]. In contrast, some studies have successfully employed non-invasive physical methods to enhance intracellular delivery of siRNA. For example, Du et al. recently demonstrated that simultaneous administration of low intensity ultrasound or 15-20% microbubbles can safely enhance the delivery efficiency of siRNA-loaded polymeric nanocarriers to rat RPE-J cells in vitro. It is likely that a combination of approaches will need to be tested to determine the optimal strategy for endosomal escape for ocular siRNA delivery.
Development of a ‘universal’ nanocarrier for ocular siRNA delivery
Literature review of ocular siRNA nanocarrier delivery
Implications for ocular diseases
IκB kinase beta (IKKβ)
Cationic nano-copolymers CS-g-(PEI-b-mPEG)
Glaucoma filtration surgery
Marked reduction in subconjuctival scarring with siRNA treatment in monkeys with trabeculectomy; higher blebs with siRNA compared to PBS treatment; less fibrosis and less destruction of local tissue in siRNA-treated eyes
Improved surgical outcome in glaucoma filtration surgery (less scarring)
IκB kinase beta (IKKβ)
Cationic nano-copolymers CS-g-(PEI-b-mPEG)
Glaucoma filtration surgery
In vitro transfection
Downregulation of IKKβ at the mRNA and protein levels; nuclear factor-κB (NF-κB) inhibited in human Tenon’s capsule fibroblasts
Decreased scar formation following glaucoma filtration surgery
PEGylated liposome- protamine- hyaluronic acid nanoparticles (PEG-LPH-NP)
Human RPE cells (ARPE19) and rats
Reduced laser-induced CNV area in rats by PEG-LPH-NP-S nanoparticles (anti-VEGFR1 siRNA) compared with naked siRNA and PEG-LPH-NP (negative siRNA); downregulated VEGFR1 expression in human RPE cells with siRNA compared to naked siRNA and control group; no significant retinal toxicity
Delivery of siRNA to decrease CNV with low toxicity
Non-specific commercial siRNA
Transit- TKO transfection reagent
Combination of siRNA with Transit - TKO transfection reagent penetrated through the inner limiting membrane into the retina and accumulated in ganglion cell layer
Uniform delivery to retinal through intravitreal injections of siRNA using commercial reagents
Moreover, the use of a nanocarrier allows for the control of immune stimulation. Kleinman et al. have shown that siRNA can directly mediate CNV suppression in vivo via a non-RNAi mediated mechanism involving cell-surface receptor TLR-3. A therapeutic siRNA shielded from the ocular environment can perhaps avoid such immune stimulation effects of siRNA. However, in some cases, it might be desirable to induce a potentially beneficial immune stimulation effect such as angiogenesis suppression. Given the versatility of nanocarrier systems, it is likely possible to design a carrier that exposes chemically modified, stabilized siRNA to ocular fluids to mediate innate immune stimulation and trigger the TLR-3 pathway for angiogenesis suppression.
The proposed four-component nanocarrier system provides a customizable platform for the development of a ‘smart’ drug delivery system that can be engineered to enhance endosomal escape, control siRNA release intracellularly and manipulate the innate immune response. Particularly, a core-shell nanocarrier structure allows for the incorporation of specific endosomal escape strategies, which can be activated upon endocytosis. For example, the core and shell components can be joined with a cleavable linker that is sensitive to endolysosomal stimuli such as acidic pH and acid-activated proteases. This design effectively allows for the de-shielding of the nanocarrier core, containing siRNA, to induce endosomal destabilization, or to directly traverse the endosomal membrane if the core has a hydrophobic composition. The reducing environment of the cytosol can also be used to further stimulate the dissociation of siRNA from the nanocarrier core via the incorporation of disulphide bonds. The first successful systemic delivery of siRNA via a targeted nanocarrier in humans serves to confirm these important parameters in nanocarrier design.
Conclusions and future directions
Given that we currently lack an ideal siRNA delivery system for ocular disorders, it is instructive to consider the nucleotide delivery strategies found in nature. For example, viruses are essentially targeted biological nanocarriers for the local or systemic delivery of nucleic acids, known to be the causative agents of various human diseases. A virion is indeed a smart nanocarrier, with several key features: environmental stability, monodispersity, bioresponsiveness, biodegradability, immune modulation properties, endosomal escape capabilities, intracellular replicative capacity, and targeted and localized DNA/RNA intracellular delivery to specific cells for controlling gene expression. To this extent, Breitbach et al. have recently shown that a modified oncolytic pox virus administered intravenously in human subjects can selectively target cancer cells in solid tumors, without any observable clinical effects on normal cells. This 300 nm enveloped virus delivered ds-DNA to target cells in a dose-dependent manner, similar to that observed in the recent Phase I clinical trial with siRNA-nanocarrier technology. A nature-inspired nanocarrier design can potentially provide structural insights into developing the optimal solutions to some of the major barriers in ocular and systemic siRNA delivery.
Many groups have employed ‘smart’ nanocarriers or ‘synthetic viruses’ that mimic isolated aspects of viral nucleotide delivery with varying degrees of success. For example, Hu et al. developed a pH-responsive core-shell nanocarrier designed to release various cargos including proteins, viral particles and siRNA under endosomal acidification. However, most of these single-stimuli responsive nanocarriers are focused on either drug delivery or for diagnostic purposes (imaging and detection), without the ability to combine such useful features. Although multiple stimuli-responsive nucleotide delivery systems are currently under development to address this challenge, a general strategy for intracellular nucleotide delivery has not yet been established. This may be due to the fact that nucleotide delivery systems vary greatly in their composition, such that combining beneficial features of two different nucleotide delivery systems into a hybrid system may not always be possible. In designing a nucleotide delivery system, it is instructive to note that viruses sequentially deploy specific strategies to overcome each barrier at the tissue and cellular level for successful intracellular nucleotide delivery. It follows that any clinically viable nucleotide delivery system will have to take into account the common barriers to siRNA delivery and incorporate specific strategies to overcome each of these barriers, while being flexible enough to combine features that can be adapted to several ocular conditions.
We envision that the ultimate ocular siRNA delivery system would incorporate a combination of nature-inspired desirable features: a biodegradable, multiple stimuli-responsive nanocarrier for controlled and localized siRNA release targeted to specific cell types for manipulating gene expression of specific genes. When combined with a drug delivery device, such a ‘smart’ nucleotide delivery system would not only address the current challenges of ocular siRNA delivery, with improved biodistribution, bioavailability and reduced toxicity, but also improve therapeutic outcomes for the patient.
No information to share.
Age-related macular degeneration
Blood aqueous barrier
Blood retinal barrier
Double stranded RNAs
Ethylene vinyl acetate
US Food and Drug Administration
Protein kinase R
RNA-induced silencing complex
Retinal pigment epithelium
Small interfering RNA
HIV transactivator of transcription
HIV-1 TAR RNA-binding protein
Vascular endothelial growth factor-A
Vascular endothelial growth factor receptor-1.
We would like to thank Prof. Mark Eiteman and the Journal of Biological Engineering for generously waiving the manuscript publication fees.
- Duvvuri S, Majumdar S, Mitra AK: Drug delivery to the retina: challenges and opportunities. Expert Opin Biol Ther 2003, 3: 45-56. 10.1517/14712522.214.171.124View ArticleGoogle Scholar
- Del Amo EM, Urtti A: Current and future ophthalmic drug delivery systems. A shift to the posterior segment. Drug DiscovToday 2008, 13: 135-143.Google Scholar
- Novack GD: Ophthalmic drug delivery: development and regulatory considerations. ClinPharmacolTher 2009, 85: 539-543.Google Scholar
- Urtti A: Challenges and obstacles of ocular pharmacokinetics and drug delivery. Adv Drug Deliv Rev 2006, 58: 1131-1135. 10.1016/j.addr.2006.07.027View ArticleGoogle Scholar
- Edelhauser HF, Rowe-Rendleman CL, Robinson MR, Dawson DG, Chader GJ, Grossniklaus HE, Rittenhouse KD, Wilson CG, Weber DA, Kuppermann BD, et al.: Ophthalmic drug delivery systems for the treatment of retinal diseases: basic research to clinical applications. Invest Ophthalmol Vis Sci 2010, 51: 5403-5420. 10.1167/iovs.10-5392View ArticleGoogle Scholar
- Kang Derwent JJ, Mieler WF: Thermoresponsive hydrogels as a new ocular drug delivery platform to the posterior segment of the eye. TransAmOphthalmolSoc 2008, 106: 206-213.Google Scholar
- Lee SS, Robinson MR: Novel drug delivery systems for retinal diseases. A review. Ophthalmic Res 2009, 41: 124-135. 10.1159/000209665View ArticleGoogle Scholar
- Kuno N, Fujii S: Biodegradable intraocular therapies for retinal disorders: progress to date. Drugs Aging 2010, 27: 117-134. 10.2165/11530970-000000000-00000View ArticleGoogle Scholar
- Campochiaro PA, Brown DM, Pearson A, Ciulla T, Boyer D, Holz FG, Tolentino M, Gupta A, Duarte L, Madreperla S, et al.: Long-term benefit of sustained-delivery fluocinolone acetonide vitreous inserts for diabetic macular edema. Ophthalmology 2011, 118: 626-635. e622 10.1016/j.ophtha.2010.12.028View ArticleGoogle Scholar
- London NJ, Chiang A, Haller JA: The dexamethasone drug delivery system: indications and evidence. Adv Ther 2011, 28: 351-366. 10.1007/s12325-011-0019-zView ArticleGoogle Scholar
- Eljarrat-Binstock E: Pe'er J, Domb AJ: New techniques for drug delivery to the posterior eye segment. Pharm Res 2010, 27: 530-543. 10.1007/s11095-009-0042-9View ArticleGoogle Scholar
- Reischl D, Zimmer A: Drug delivery of siRNA therapeutics: potentials and limits of nanosystems. Nanomedicine: Nanotechnology, Biology and Medicine 2009, 5: 8-20. 10.1016/j.nano.2008.06.001Google Scholar
- Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature 1998, 391: 806-811. 10.1038/35888View ArticleGoogle Scholar
- Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001, 411: 494-498. 10.1038/35078107View ArticleGoogle Scholar
- de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J: Interfering with disease: a progress report on siRNA-based therapeutics. Nature Rev Drug Discov 2007, 6: 443-453. 10.1038/nrd2310View ArticleGoogle Scholar
- Ahmed SU, Milner J: Basal Cancer Cell Survival Involves JNK2 Suppression of a Novel JNK1/c-Jun/Bcl-3 Apoptotic Network. PLoS One 2009, 4: e7305. 10.1371/journal.pone.0007305View ArticleGoogle Scholar
- Watts JK, Deleavey GF, Damha MJ: Chemically modified siRNA: tools and applications. Drug Discovery Today 2008, 13: 842-855. 10.1016/j.drudis.2008.05.007View ArticleGoogle Scholar
- Whitehead K, Langer R, Anderson D: Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 2009, 8: 129-138. 10.1038/nrd2742View ArticleGoogle Scholar
- Kennedy S, Wang D, Ruvkun G: A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature. 2004, 427: 645-649.Google Scholar
- Pirollo K, Chang E: Targeted delivery of small interfering RNA: approaching effective cancer therapies. Cancer Res 2008, 68: 1247-1250. 10.1158/0008-5472.CAN-07-5810View ArticleGoogle Scholar
- Mohan R, Tovey J, Sharma A, Tandon A: Gene therapy in the Cornea: 2005-present. Prog Retin Eye Res 2012, 31: 43-64. 10.1016/j.preteyeres.2011.09.001View ArticleGoogle Scholar
- Couto L, High K: Viral vector-mediated RNA interference. Curr Opin Pharmacol 2010, 10: 534-542. 10.1016/j.coph.2010.06.007View ArticleGoogle Scholar
- Wu L, Lam S, Cao H, Guan R, Hu J: Subretinal gene delivery using helper-dependent adenoviral vectors. Cell Biosci 2011, 1: 15. 10.1186/2045-3701-1-15View ArticleGoogle Scholar
- Höbel S, Koburger I, John M, Czubayko F, Hadwiger P, Vornlocher H, Aigner A: Polyethylenimine/small interfering RNA-mediated knockdown of vascular endothelial growth factor in vivo exerts anti-tumor effects synergistically with Bevacizumab. J Gene Med 2010, 12: 287-300.Google Scholar
- Hirano Y, Sakurai E, Matsubara A, Ogura Y: Suppression of ICAM-1 in retinal and choroidal endothelial cells by plasmid small-interfering RNAs in vivo. Invest Ophthalmol Vis Sci 2010, 51: 508-515. 10.1167/iovs.09-3457View ArticleGoogle Scholar
- Conley S, Naash M: Nanoparticles for retinal gene therapy. Prog Retin Eye Res 2010, 29: 376-397. 10.1016/j.preteyeres.2010.04.004View ArticleGoogle Scholar
- Khar R, Jain G, Warsi M, Mallick N, Akhter S, Pathan S, Ahmad F: Nano-vectors for the Ocular Delivery of Nucleic Acid-based Therapeutics. Indian J Pharm Sci 2010, 72: 675-688. 10.4103/0250-474X.84575View ArticleGoogle Scholar
- Naik R, Mukhopadhyay A, Ganguli M: Gene delivery to the retina: focus on non-viral approaches. Drug Discov Today 2009, 14: 306-315. 10.1016/j.drudis.2008.09.012View ArticleGoogle Scholar
- Boussif O: Lezoualc'h F, Zanta M, Mergny M, Scherman D, Demeneix B, Behr J: A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. PNAS 1995, 92: 7297-7301. 10.1073/pnas.92.16.7297View ArticleGoogle Scholar
- Grayson A, Doody A, Putnam D: Biophysical and Structural Characterization of Polyethylenimine-Mediated siRNA Delivery in Vitro. Pharm Res 2006, 23: 1868-1876. 10.1007/s11095-006-9009-2View ArticleGoogle Scholar
- Kim B, Tang Q, Biswas P, Xu J, Schiffelers R, Xie F, Ansari A, Scaria P, Woodle M, Lu P, Rouse B: Inhibition of ocular angiogenesis by siRNA targeting vascular endothelial growth factor pathway genes: therapeutic strategy for herpetic stromal keratitis. Am J Pathol 2004, 165: 2177-2185. 10.1016/S0002-9440(10)63267-1View ArticleGoogle Scholar
- Günther M, Lipka J, Malek A, Gutsch D, Kreyling W, Aigner A: Polyethylenimines for RNAi-mediated gene targeting in vivo and siRNA delivery to the lung. Eur J Pharm Biopharm 2011, 77: 438-449. 10.1016/j.ejpb.2010.11.007View ArticleGoogle Scholar
- Duan Y, Guan X, Ge J, Quan D, Zhuo Y, Ye H, Shao T: Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro. Mol Vis 2008, 14: 2616-2628.Google Scholar
- Ye H, Qian Y, Lin M, Duan Y, Sun X, Zhuo Y, Ge J: Cationic nano-copolymers mediated IKKβ targeting siRNA to modulate wound healing in a monkey model of glaucoma filtration surgery. Mol Vis 2010, 16: 2502-2510.Google Scholar
- Soriano P, Dijkstra J, Legrand A, Spanjer H, Londos-Gagliardi D, Roerdink F, Scherphof G, Nicolau C: Targeted and nontargeted liposomes for in vivo transfer to rat liver cells of a plasmid containing the preproinsulin I gene. PNAS 1983, 80: 7128-7131. 10.1073/pnas.80.23.7128View ArticleGoogle Scholar
- Liu H, Liu Y, Ma Z, Wang J, Zhang Q: A Lipid Nanoparticle System Improves siRNA Efficacy in RPE Cells and a Laser-Induced Murine CNV Model. Invest Ophthalmol Vis Sci 2011, 52: 4789-4794. 10.1167/iovs.10-5891View ArticleGoogle Scholar
- Zhang Y, Li H, Sun J, Gao J, Liu W, Li B, Guo Y, Chen J: DC-Chol/DOPE cationic liposomes: a comparative study of the influence factors on plasmid pDNA and siRNA gene delivery. Int J Pharm 2010, 390: 198-207. 10.1016/j.ijpharm.2010.01.035View ArticleGoogle Scholar
- Lochmann D, Weyermann J, Georgens C, Prassl R, Zimmer A: Albumin-protamine-oligonucleotide nanoparticles as a new antisense delivery system. Part 1: Physicochemical characterization. Eur J Pharm Biopharm 2005, 59: 419-429. 10.1016/j.ejpb.2004.04.001View ArticleGoogle Scholar
- Johnson L, Cashman S, Kumar-Singh R: Cell-penetrating peptide for enhanced delivery of nucleic acids and drugs to ocular tissues including retina and cornea. Mol Ther 2008, 16: 107-114. 10.1038/sj.mt.6300324View ArticleGoogle Scholar
- Laufer S, Restle T: Peptide-Mediated Cellular Delivery of Oligonucleotide-Based Therapeutics In Vitro: Quantitative Evaluation of Overall Efficacy Employing Easy to Handle Reporter Systems. Curr Pharm Des 2008, 14: 3637-3655. 10.2174/138161208786898806View ArticleGoogle Scholar
- Yuan X, Naguib S, Wu Z: Recent advances of siRNA delivery by nanoparticles. Expert Opin Drug Deliv 2011, 8: 521-536. 10.1517/17425247.2011.559223View ArticleGoogle Scholar
- Marano R, Wimmer N, Kearns P, Thomas B, Toth I, Brankov M, Rakoczy P: Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide. Exp Eye Res 2004, 79: 525-535. 10.1016/j.exer.2004.06.023View ArticleGoogle Scholar
- Marano R, Toth I, Wimmer N, Brankov M, Rakoczy P: Dendrimer delivery of an anti-VEGF oligonucleotide into the eye: a long-term study into inhibition of laser-induced CNV, distribution, uptake and toxicity. Gene Ther 2005, 12: 1544-1550. 10.1038/sj.gt.3302579View ArticleGoogle Scholar
- Agrawal A, Min D, Singh N, Zhu H, Bhatia S: Functional delivery of siRNA in mice using dendriworms. ACS Nano 2009, 3: 2495-2504. 10.1021/nn900201eView ArticleGoogle Scholar
- Han L, Zhang A, Wang H, Pu P, Jiang X, Kang C, Chang J: Tat-BMPs-PAMAM conjugates enhance therapeutic effect of small interference RNA on U251 glioma cells in vitro and in vivo. Hum Gene Ther 2010, 21: 417-426. 10.1089/hum.2009.087View ArticleGoogle Scholar
- A Dose Escalation Trial of an Intravitreal Injection of Sirna-027 in Patients With Subfoveal Choroidal Neovascularization (CNV) Secondary to Age-Related Macular Degeneration (AMD). [http://clinicaltrials.gov/ct2/show/NCT00363714] 
- A Study Using Intravitreal Injections of a Small Interfering RNA in Patients With Age-Related Macular Degeneration. [http://clinicaltrials.gov/ct2/show/NCT00395057] 
- Kaiser P, Symons R, Shah S, Quinlan E, Nguyen Q: RNAi-based treatment for neovascular age-related macular degeneration by Sirna-027. Am J Ophthalmol 2010, 150: 33-39. 10.1016/j.ajo.2010.02.006View ArticleGoogle Scholar
- Safety & Efficacy Study Evaluating the Combination of Bevasiranib & Lucentis Therapy in Wet AMD (COBALT). [http://clinicaltrials.gov/ct2/show/NCT00499590] 
- Phase II Open Label Multicenter, Prospective, Randomized, Age Related Macular Degeneration, Comparator Controlled Study Evaluating PF-04523655 Versus Ranibizumab In The Treatment Of Subjects With Choroidal Neovascularization (MONET Study). [http://www.clinicaltrials.gov/ct2/show/NCT00713518?term=quark&rank=7] 
- Prospective, Randomized, Multi-Center, Comparator Study Evaluating Efficacy and Safety of PF-04523655 Versus Laser in Subjects With Diabetic Macular Edema (DEGAS). [http://www.clinicaltrials.gov/ct2/show/NCT00701181?term=degas&rank=1] 
- Development pipeline: PF-655. [http://www.quarkpharma.com/qbi-en/products/53/] 
- Safety Study of a Single IVT Injection of QPI-1007 in Chronic Optic Nerve Atrophy and Recent Onset NAION Patients. [http://clinicaltrials.gov/ct2/show/NCT01064505] 
- Tolerance and Effect on Intraocular Pressure After Administration of SYL040012. [http://clinicaltrials.gov/ct2/show/NCT00990743] 
- Tiemann K, Rossi J: RNAi-based therapeutics-current status, challenges and prospects. EMBO Mol Med 2009, 1: 142-151. 10.1002/emmm.200900023View ArticleGoogle Scholar
- Morrissey D, Blanchard K, Shaw L, Jensen K, Lockridge J, Dickinson B, McSwiggen J, Vargeese C, Bowman K, Shaffer C, et al.: Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication. Hepatology 2005, 41: 1349-1356. 10.1002/hep.20702View ArticleGoogle Scholar
- Pipeline: Development Programs. [http://www.alnylam.com/Programs-and-Pipeline/index.php] 
- Judge AD, Sood V, Shaw JR, Fang D, McClintock K, MacLachlan I: Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 2005, 23: 457-462. 10.1038/nbt1081View ArticleGoogle Scholar
- Prakash T, Allerson C, Dande P, Vickers T, Sioufi N, Jarres R, Baker B, Swayze E, Griffey R, Bhat B: Positional effect of chemical modifications on short interference RNA activity in mammalian cells. J Med Chem 2005, 48: 4247-4253. 10.1021/jm050044oView ArticleGoogle Scholar
- Czauderna F, Fechtner M, Dames S, Aygün H, Klippel A, Pronk G, Giese K, Kaufmann J: Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res 2003, 31: 2705-2716. 10.1093/nar/gkg393View ArticleGoogle Scholar
- Morrissey D, Lockridge J, Shaw L, Blanchard K, Jensen K, Breen W, Hartsough K, Machemer L, Radka S, Jadhav V, et al.: Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nature Biotechnol 2005, 23: 1002-1007. 10.1038/nbt1122View ArticleGoogle Scholar
- Samuel-Abraham S, Leonard J: Staying on message: design principles for controlling nonspecific responses to siRNA. FEBS J 2010, 277: 4828-4836. 10.1111/j.1742-4658.2010.07905.xView ArticleGoogle Scholar
- Leuschner P, Ameres S, Kueng S, Martinez J: Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep 2006, 7: 314-320. 10.1038/sj.embor.7400637View ArticleGoogle Scholar
- Hall AH, Wan J, Shaughnessy EE, Ramsay Shaw B, Alexander KA: RNA interference using boranophosphate siRNAs: structure-activity relationships. Nucleic Acids Res 2004, 32: 5991-6000. 10.1093/nar/gkh936View ArticleGoogle Scholar
- Choung S, Kim Y, Kim S, Park HO, Choi YC: Chemical modification of siRNAs to improve serum stability without loss of efficacy. Biochem Biophys Res Commun 2006, 342: 919-927. 10.1016/j.bbrc.2006.02.049View ArticleGoogle Scholar
- Shim M, Kwon Y: Efficient and targeted delivery of siRNA in vivo. FEBS J 2010, 277: 4814-4827. 10.1111/j.1742-4658.2010.07904.xView ArticleGoogle Scholar
- Overhoff M, Sczakiel G: Phosphorothioate-stimulated uptake of short interfering RNA by human cells. EMBO Rep 2005, 6: 1176-1181. 10.1038/sj.embor.7400535View ArticleGoogle Scholar
- Behlke M: Chemical modification of siRNAs for in vivo use. Oligonucleotides 2008, 18: 305-319. 10.1089/oli.2008.0164View ArticleGoogle Scholar
- Jackson A, Burchard J, Leake D, Reynolds A, Schelter J, Guo J, Linsley P: Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing. RNA 2006, 12: 1197-1205. 10.1261/rna.30706View ArticleGoogle Scholar
- Minks M, West D, Benvin S, Baglioni C: Structural requirements of double-stranded RNA for the activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells. J Biol Chem 1979, 254: 10180-10183.Google Scholar
- Marques JT, Williams BRG: Activation of the mammalian immune system by siRNAs. Nat Biotechnol 2005, 23: 1399-1405. 10.1038/nbt1161View ArticleGoogle Scholar
- Kleinman ME, Yamada K, Takeda A, Chandrasekaran V, Nozaki M, Baffi JZ, Albuquerque RJC, Yamasaki S, Itaya M, Pan Y, et al.: Sequence- and target-independent angiogenesis suppression by siRNA via TLR3. Nature 2008, 452: 591-597. 10.1038/nature06765View ArticleGoogle Scholar
- Judge A, MacLachlan I: Overcoming the innate immune response to small interfering RNA. Human Gene Therapy 2008, 19: 111-124. 10.1089/hum.2007.179View ArticleGoogle Scholar
- Morrissey DV: Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nature Biotechnol 2005, 23: 1002-1007. 10.1038/nbt1122View ArticleGoogle Scholar
- Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Cavet G, Linsley PS: Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 2003, 21: 635-637. 10.1038/nbt831View ArticleGoogle Scholar
- Walton S, Wu M, Gredell J, Chan C: Designing highly active siRNAs for therapeutic applications. FEBS J 2010, 277: 4806-4813. 10.1111/j.1742-4658.2010.07903.xView ArticleGoogle Scholar
- Zuhorn IS, Kalicharan R, Hoekstra D: Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis. J Biol Chem 2002, 277: 18021-18028. 10.1074/jbc.M111257200View ArticleGoogle Scholar
- Torchilin V: Cell penetrating peptide-modified pharmaceutical nanocarriers for intracellular drug and gene delivery. Biopolymers 2008, 90: 604-610. 10.1002/bip.20989View ArticleGoogle Scholar
- Petros R, DeSimone J: Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov 2010, 9: 615-627. 10.1038/nrd2591View ArticleGoogle Scholar
- Rejman J, Oberle V, Zuhorn IS, Hoekstra D: Size-dependent internalization of particles via the pathways of clathrin-and caveolae-mediated endocytosis. Biochem J 2004, 377: 159-169. 10.1042/BJ20031253View ArticleGoogle Scholar
- Fisher D, Ahlemeyer Y, Krieglstein B, Kissel T: In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials 2003, 24: 1121-1131. 10.1016/S0142-9612(02)00445-3View ArticleGoogle Scholar
- Kim BYS, Jiang W, Oreopoulos J, Yip CM, Rutka JT, Chan WCW: Biodegradable Quantum Dot Nanocomposites Enable Live Cell Labeling and Imaging of Cytoplasmic Targets. Nano Letters 2008, 8: 3887-3892. 10.1021/nl802311tView ArticleGoogle Scholar
- Rajendran L, Knölker H, Simons K: Subcellular targeting strategies for drug design and delivery. Nat Rev Drug Discov 2010, 9: 29-42. 10.1038/nrd2897View ArticleGoogle Scholar
- Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K, Sugiura Y: Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem 2001, 276: 5836-5840.Google Scholar
- Ruan G, Agrawal A, Marcus AI, Nie S: Imaging and Tracking of Tat Peptide-Conjugated Quantum Dots in Living Cells: New Insights into Nanoparticle Uptake, Intracellular Transport, and Vesicle Shedding. J Am Chem Soc 2007, 129: 14759-14766. 10.1021/ja074936kView ArticleGoogle Scholar
- Derossi D, Joliot A, Chassaing G, Prochiantz A: The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem 1994, 269: 10444-10450.Google Scholar
- Duan H, Nie S: Cell-penetrating quantum dots based on multivalent and endosome-disrupting surface coatings. J Am Chem Soc 2007, 129: 3333-3338. 10.1021/ja068158sView ArticleGoogle Scholar
- Thomas M, Klibanov A: Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells. PNAS 2002, 99: 14640-14645. 10.1073/pnas.192581499View ArticleGoogle Scholar
- Gusachenko Simonova O, Kravchuk Y, Konevets D, Silnikov V, Vlassov VV, Zenkova MA: Transfection Efficiency of 25-kDa PEICholesterol Conjugates with Different Levels of Modification. J Biomater Sci Polym Ed 2009, 20: 1091-1110. 10.1163/156856209X444448View ArticleGoogle Scholar
- Bouxsein N, McAllister C, Ewert K, Samuel C, Safinya C: Structure and gene silencing activities of monovalent and pentavalent cationic lipid vectors complexed with siRNA. Biochemistry 2007, 46: 4785-4792. 10.1021/bi062138lView ArticleGoogle Scholar
- Leal C, Bouxsein N, Ewert K, Safinya C: Highly efficient gene silencing activity of siRNA embedded in a nanostructured gyroid cubic lipid matrix. J Am Chem Soc 2010, 132: 16841-16847. 10.1021/ja1059763View ArticleGoogle Scholar
- Leal C, Ewert K, Shirazi R, Bouxsein N, Safinya C: Nanogyroids incorporating multivalent lipids: enhanced membrane charge density and pore forming ability for gene silencing. Langmuir 2011, 27: 7691-7697. 10.1021/la200679xView ArticleGoogle Scholar
- Du J, Shi Q, Sun Y, Liu P, Zhu M, Du L, Duan Y: Enhanced delivery of monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly l-lysine nanoparticles loading platelet-derived growth factor BB small interfering RNA by ultrasound and/or microbubbles to rat retinal pigment epithelium cells. J Gene Med 2011, 13: 312-323. 10.1002/jgm.1574View ArticleGoogle Scholar
- Turchinovich A, Zoidl G, Dermietzel R: Non-viral siRNA delivery into the mouse retina in vivo. BMC Ophthalmol 2010, 10: 25. 10.1186/1471-2415-10-25View ArticleGoogle Scholar
- Aggarwal P, Hall J, McLeland C, Dobrovolskaia A, McNeil S: Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy. Adv Drug Deliv Rev 2009, 61: 428-437. 10.1016/j.addr.2009.03.009View ArticleGoogle Scholar
- Gref R, Minamitake Y, Peracchia M, Trubetskoy V, Torchilin V, Langer R: Biodegradable long-circulating polymeric nanospheres. Science 1994, 263: 1600-1603. 10.1126/science.8128245View ArticleGoogle Scholar
- Wong C, Stylianopoulos T, Cui J, Martin J, Fukumura D: Multistage nanoparticle delivery system for deep penetration into tumor tissue. PNAS 2011, 108: 2426-2431. 10.1073/pnas.1018382108View ArticleGoogle Scholar
- Gratton S, Ropp P, Pohlhaus P, Luft J, Madden V, Napier M, DeSimone J: The effect of particle design on cellular internalization pathways. PNAS 2008, 105: 11613-11618. 10.1073/pnas.0801763105View ArticleGoogle Scholar
- Yokoe J, Sakuragi S, Yamamoto K, Teragaki T, Ogawara K, Higaki K, Katayama N, Kai T, Sato M, Kimura T: Albumin-conjugated PEG liposome enhances tumor distribution of liposomal doxorubicin in rats. Int J Pharm 2008, 353: 28-34. 10.1016/j.ijpharm.2007.11.008View ArticleGoogle Scholar
- Furumoto K, Yokoe J, Ogawara K, Amano S, Takaguchi M, Higaki K, Kai T, Kimura T: Effect of coupling of albumin onto surface of PEG liposome on its in vivo disposition. Int J Pharm 2007, 329: 110-116. 10.1016/j.ijpharm.2006.08.026View ArticleGoogle Scholar
- Davis M, Zuckerman J, Choi C, Seligson D, Ribas A: Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 2010, 464: 1067-1070. 10.1038/nature08956View ArticleGoogle Scholar
- Breitbach C, Burke J, Jonker D, Stephenson J, Haas A, Kirn D: Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature 2011, 477: 99-102. 10.1038/nature10358View ArticleGoogle Scholar
- Hu Y, Atukorale P, Lu J, Moon J, Um S, Cho E, Wang Y, Chen J, Irvine D: Cytosolic delivery mediated via electrostatic surface binding of protein, virus, or siRNA cargos to pH-responsive core-shell gel particles. Biomacromolecules 2009, 10: 756-765. 10.1021/bm801199zView ArticleGoogle Scholar
- You J, Almeda D, Ye G, Auguste D: Bioresponsive matrices in drug delivery. J Biol Eng 2010., 4:Google Scholar
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