Peptide-targeted delivery of siRNA may offer a combination of the strong silencing capacity of RNA interference and the tissue selectivity lacking in naked oligonucleotides. Attaching a cell-penetrating peptide with an affinity for a specific receptor may direct the siRNA to disease relevant cells, avoiding non-target tissues. The strategy is, however, hampered by three major drawbacks: competition with endogenous ligands, inconstant expression of the target, and proteolytic instability of the peptides. These problems frequently result in a limited amount of delivered siRNA actually reaching the functional compartment. Thus, a potentially useful therapeutic agent becomes a victim of pharmacokinetics.
siRNA is extremely potent – as few as single molecules can quench target mRNA expression with catalytic efficiency – but that intrinsic activity is of little use if the oligonucleotide never arrives at the cytosol. The challenge is how to bridge the gap between the test-tube potency of perfectly base-paired duplexes and the realities of systemic delivery where nucleases, immune sensors and biological membranes combine to degrade or eject the payload before it can reach the RNA-induced silencing complex. Current platforms work in the liver or other locally accessible compartments but are not up to the task of reaching extra-hepatic tissues with sufficient selectivity and durability. Delivery is the gap that must be closed if siRNA is to move from laboratory curiosity to broadly applicable therapeutic modality.
Fig. 1 Delivery paradigms for siRNA therapeutics based on the clinical requirements: (a) drug modification, (b) environment modification, and (c) drug delivery system. 1,5
siRNA duplexes are highly catalytically potent but their polyanionic nature and high molecular weight makes it impossible for them to cross biological membranes. On systemic administration, they are subjected to nuclease activity in the serum and in endosomal compartment, which limits the half-life of naked siRNA to a few minutes. Protected by complexation, the endosomal escape of siRNA into cytosol is poor, with less than 1% of the internalized drug being able to access the RISC. The dissociation between the in-vitro potency and in-vivo access means that delivery rather than target engagement is the rate-limiting step for therapeutic potency. No matter how high the potency of a particular siRNA molecule, without a vehicle to deliver it across the biological barriers, the siRNA catalytic potency would be for nothing. Lipid nanoparticles and polymer complexes have increased the stability and residence time of RNAi therapeutics in circulation, but their biodistribution is broad with high accumulation in liver and spleen but not in the tissues of therapeutic interest. Attempts to use peptide-mediated targeting to direct uptake through receptor-mediated endocytosis have also been made but have been similarly limited by serum proteolysis, endosomal entrapment and competition for receptors. For these reasons, delivery is the major hurdle in the clinical translation of siRNA drugs.
Systemically administered naked or formulated siRNA is largely sequestered in fenestrated organs such as liver, kidney and spleen by mechanisms of size-based filtration and passive phagocytosis. This in vivo organ tropism greatly reduces utility for extra-hepatic indications and necessitates dose reduction to mitigate hepatotoxicity, which can compromise efficacy at the desired site of action. Tissue selectivity can only be achieved by molecular filters that actively discriminate between healthy and diseased cells, a function that passive carriers are incapable of performing. Current delivery platforms lack fine-grained molecular recognition and non-selectively deliver siRNA to any cell with an endocytic capacity. The lack of ligand-mediated targeting also precludes selectivity of gene silencing in healthy tissues, which can give rise to dose-limiting toxicities. Peptide-mediated targeting is one approach to improve this, but receptor expression heterogeneity, competition with endogenous ligands, and variable internalization kinetics make it difficult to engineer and validate true cell-type selectivity in vivo.
Table 1 Peptide-Mediated siRNA Delivery: Mechanisms and Limitations
| Feature | Mechanism | Challenge | Countermeasure |
| Receptor binding | Specific recognition | Competition with endogenous ligands | Allosteric epitope design |
| Endosomal escape | pH-sensitive fusion | Low efficiency | Histidine-rich spacers |
| Biodistribution | Active targeting | Hepatic clearance dominance | Transient albumin shield |
| Stability | Protease resistance | Rapid degradation | Cyclization, D-amino acids |
| Specificity | Target antigen density | Normal tissue expression | Multi-epitope AND logic |
Peptide-mediated siRNA delivery is a cell-targeting strategy for which siRNA is complexed with short synthetic peptides to deliver siRNA to certain cell types. Peptides can be used to target receptors on the cell membrane and promote cellular uptake by receptor-mediated endocytosis. Some peptides have membrane permeabilization properties which allow for translocation of the siRNA across the membrane and into the cytoplasm. Directing transport to specific tissues by the addition of molecular recognition elements may improve delivery specificity and reduce off-target effects. There are two major strategies for peptide-mediated delivery: direct covalent conjugation and non-covalent complexation. In the former case, the siRNA is covalently bound to a targeting peptide, while the latter involves the formation of peptide-siRNA complexes through electrostatic or hydrophobic interactions.
Conjugating the siRNA directly to the targeting peptide in a stoichiometric and chemically well-defined manner can be achieved by covalent linkage. Amide or disulfide bonds between the 3′ or 5′ end of the siRNA strand and N-terminus or a side-chain of the peptide are commonly used for such conjugates. Such constructs have increased serum stability and a predictable pharmacokinetic profile due to the absence of premature dissociation of the nucleic acid moiety. On the other hand, the siRNA is a bulky, highly anionic species that can sterically hinder the interaction of the peptide with its target, reducing affinity or even completely abolishing targeting. The covalent linkage is also rigid, which can also preclude endosomal escape. Thus, cleavable linkers that release the siRNA payload upon reduction or enzymatic cleavage inside the target cell are often incorporated. In this concept, cationic peptides form a complex with anionic siRNA by charge-neutralisation, yielding nanoscale particles that are held together by non-covalent forces. This particle assembly is dynamic and can readily be disassembled upon increased ionic strength or competitive displacement, which can be exploited to promote cytosolic release. In practice, these complexes are susceptible to dissociation in serum, and albumin and other polyanions can 'strip' the siRNA off the peptide leading to a loss of cargo and bioavailability. Stability of such complexes can be improved by cross-linking or by including hydrophobic anchors that promote particle condensation, but such modifications can increase particle size above the renal filtration cut-off size, and change biodistribution in an undesirable way.
Targeting peptides contain motifs such as RGD, bombesin analogues or somatostatin receptor ligands that bind to overexpressed cell-surface receptors on cancer cells. Receptor binding facilitates clathrin-mediated or caveolae-mediated endocytosis, leading to internalization of the endosome-entrapped siRNA cargo. Active targeting leads to a high local concentration of the siRNA at the cell membrane, while preventing sequestration in non-specific tissue sinks. The choice of target receptor is critical to defining the overall tissue tropism, with receptors that are less expressed on healthy tissue affording improved selectivity. Tissue distribution is also determined by the relative abundance of the target receptor relative to endogenous ligands, which may saturate available binding sites. Consequently, uptake is potentially improved by optimizing the peptide for high receptor affinity, or by carefully timing administration to a period of low endogenous ligand abundance. In addition to uptake, the post-binding trafficking and receptor kinetics also affect the fate of the siRNA. Fast recycling receptors (integrins) may recycle the conjugate to the cell surface before endosomal escape, while trafficking to the lysosomes can provide extended intracellular residence time for processing. Peptides that induce receptor clustering, or that are fused to endosomal membrane perturbants, can promote release into the cytosol. Incorporation of pH-responsive fusogenic peptides (e.g. HA2 analogues) that can undergo a conformational transition in the acidic endosome can disrupt endosomal membranes and allow release of the siRNA into the cytosol.
The use of targeting peptides changes the siRNA delivery strategy from passive, bulk drug distribution to active, receptor-mediated siRNA delivery. Targeting peptides provide a molecular address that can direct the nucleic acid payload to specific cell types while minimizing distribution to off-target organs. Active delivery circumvents the "lottery" of enhanced permeability and retention, instead relying on cell-surface recognition to selectively accumulate the siRNA at the site of disease with high precision. The chemistry of peptide synthesis is modular, allowing for rapid re-design if the siRNA sequence, chemical modifications, or formulation platform are changed, providing a plug-and-play platform with the opportunity to further optimize for each therapeutic target. Targeting via peptides may also allow a lower total systemic dose to be used to achieve a biologically active dose at the target site, which could lower potential toxicity while increasing the therapeutic index. This is of particular importance for chronic or repeat-dose treatment regimens, where cumulative exposure is a concern.
In contrast to passive absorption, targeting peptides home to cell-surface receptors that are differentially expressed between diseased and healthy tissue, converting the delivery of siRNA from a random process to a molecularly addressed event. Active recognition localizes and enriches the siRNA at the desired site, decreasing off-target delivery to liver, spleen, or kidney, which serve as sites of accumulation for lipid-based carriers. Targeting peptides may achieve cell-type selectivity by binding to receptors that are overexpressed on tumor cells or activated endothelium, an option that is not available to passive carriers. Focusing the payload in this way decreases the therapeutic window and circumvents the dose-limiting toxicities associated with activating RNAi on healthy tissue. Peptides are cleared renally and do not build up in phagocytic compartments, thus background in healthy tissues is limited. Only those cells that express the target receptor will internalize the siRNA, thereby sparing surrounding tissue from off-target gene knockdown. When the target receptor is upregulated as a result of disease, this affords an even higher degree of selectivity because the targeting gate is closed as the disease pathology subsides.
Adaptability to various siRNA architectures is possible due to the diversity of chemical strategies available in peptide conjugation: phosphorothioate backbones, 2′-O-methyl modifications, or even self-delivering siRNA architectures are all compatible with a common conjugation strategy. Additionally, the covalent attachment through reducible disulfides or non-covalent assembly through electrostatic complexation each provide adjustable stability, permitting the chemical identity of the payload to be matched with desired release kinetics. The same can be said for the architecture itself: linear peptides, cyclic scaffolds, or dendritic multimers each have their own synthetic and biological advantages, and enable one to easily iterate the design without having to redevelop the overall vector platform. By tuning the length, charge density, and valency of the peptide, it is possible to achieve the desired particle size, serum stability, and endosomal escape efficiency. The incorporation of pH-sensitive fusogenic domains or self-immolative spacers can further enable conditional cargo release, creating a stimulus-responsive delivery system from a generic peptide carrier and promoting unpacking of siRNA only in the acidic environment of endosomes or upon receptor clustering.
Active targeting allows higher intracellular levels of siRNA per administered dose relative to passive accumulation, and thus lower systemic doses while maintaining an effective gene silencing. In tumor xenograft models, peptide-mediated delivery has achieved gene silencing and tumor growth suppression at doses ten-fold lower than lipid nanoparticle delivery. This provides not only lower manufacturing cost and less off-target stimulation of the immune system but also a much-needed reduction in the size of the dose for subsequent in-human use. The key to this efficiency is that the targeting receptor is recycled. One targeting peptide can mediate multiple rounds of siRNA internalization without the need to increase the dose. Efficacy is not measured in terms of cellular uptake but in terms of the degree of target knockdown. Peptide carriers that have endosomolytic motifs also enhance the proportion of internalized siRNA that is able to reach the cytosol, and thus increase functional delivery of siRNA, and enable further dose reduction. Because peptides are eliminated renally, the free, unconjugated siRNA is excreted and does not build up in normal tissues. This increases the therapeutic window to be limited to malignant cells that are actively internalizing the conjugate and decreases the total amount of nucleic acid burden to the patient and subsequent immunogenicity and metabolic burden.
Peptide delivery of siRNA has been riddled with many different problems making it unsuitable for clinical translation. One of these problems is achieving the optimal balance in several parameters that are needed for efficient siRNA delivery. For example, the peptide used should be stable in the circulation, yet cleavable after it enters the cell to release its cargo; the peptide should be cationic enough to condense siRNA into a nanoparticle, yet its charge should not be too high, to prevent it from sticking to normal cells and tissues. In other words, this is a double-edged sword. To a certain extent, all of these problems can be achieved. However, most of the times this does not happen for several reasons: the resulting complex may be unstable and release its siRNA cargo prematurely; it may dissociate after injection in the serum; it may become trapped in endosomes and never make it to the cytosol; and the target receptors on the surface of the cells may be saturated, before reaching an efficacious dose in the cell or tissue of interest. The disease microenvironment in general is also complex, and many of the parameters involved in siRNA delivery are greatly affected by it. The proteases present in a tumor microenvironment can lead to instability, whereas there may be competing ligands or heterogeneity in receptor expression which can add to the failure of many of these preclinical siRNA delivery systems. This section focuses on the most common pitfalls associated with peptide-mediated siRNA delivery, with a particular focus on stability, endosomal entrapment, and receptor competition.
Table 2 Core challenges in peptide-siRNA delivery
| Challenge | Underlying Cause | Functional Consequence |
| Serum nuclease degradation | Exposed phosphodiester backbone | Loss of active siRNA before targeting |
| Membrane impermeability | Anionic charge, size | Minimal cellular uptake |
| Renal filtration | Sub-5 kDa hydrodynamic radius | Short circulation half-life |
| Endosomal entrapment | Trafficking to lysosomes | Cytosolic delivery failure |
| Target heterogeneity | Variable receptor density | Uneven silencing across lesions |
Linear peptides constructed from natural L-amino acids are substrates of the pan-specific serum aminopeptidases, carboxypeptidases, and chymotrypsin-like endoproteases. Recognition epitopes are cleaved within minutes of intravenous injection, and inactive cleavage products compete with native peptide-siRNA conjugates for specific receptor binding. The resulting proteolytic instability translates to a poor circulatory half-life, often much shorter than the time necessary to penetrate tumors and engage receptors. Conjugates with enhanced serum stability can be obtained by cyclization, N-methylation, and the substitution of D-amino acids, but such modifications usually lower receptor binding or increase immunogenicity, diminishing targeting fidelity. The peptide-siRNA linkage, in particular disulfide bonds, can also be cleaved by plasma thiols prior to cellular uptake, which can result in payload loss and systemic exposure of naked siRNA, which is rapidly degraded by nucleases and may cause off-target activation of immune pathways. Non-covalent peptide-siRNA complexes, held together primarily by electrostatic interactions, can also dissociate due to competition with serum proteins and polyanions such as heparan sulfate. Albumin and other serum proteins can bind cationic peptide residues and displace siRNA, subjecting it to nuclease degradation. Dissociation of the complex is concentration-dependent and becomes more pronounced after dilution in the blood, where the equilibrium shifts toward dissociated components. As a result, a significant portion of siRNA payload dissociates from the carrier before reaching target cells, which reduces functional delivery efficiency and increases off-target effects. Stabilizing components such as PEGylated linkers or polymeric backbones may be added to prevent dissociation, but these increase the molecular weight and often sterically hinder receptor binding, forcing a compromise between complex stability and targeting efficiency that is difficult to generalize across species.
Receptor-mediated endocytosis allows for effective cellular internalization of peptide-siRNA conjugates into early endosomes. The next step, known as endosomal escape, is highly inefficient. The majority (> 90%) of the siRNA payload internalized via this route becomes sequestered within endolysosomal compartments where low pH and high concentrations of proteases will degrade the contents before they have a chance to access the RISC. The uncoupling of cellular uptake from functional activity is the single largest source of poor gene silencing efficiency. Engineering peptides with fusogenic or pH-responsive motifs can help destabilize the endosomal membrane, but these modifications can also cause indiscriminate plasma membrane or lysosome disruption in healthy cells, which increases toxicity. Avoiding off-target endosomolysis has proven a particular challenge. Even in the best-case scenario where the targeting peptide successfully traffics to the endosomal system, the kinetics may be mismatched with the stability of the siRNA cargo. For example, the maturation process of late endosomes takes 30-60 minutes, within which the RNase environment will begin to degrade unprotected siRNA. In the absence of an effective escape strategy, functional delivery will be minimal and the data from uptake-focused imaging experiments will provide an artificially high positive readout. To circumvent this bottleneck, methods such as photochemical internalization and exogenous ultrasound application have been trialed to mechanically rupture endosomes, but these physical methods add clinical complexity and are not broadly applicable to all cargo. The fundamental problem remains: the vast majority of peptide carriers developed over the last two decades perform well in one arena (targeting) but poorly in the final and often most difficult intracellular release step.
Disease-targeting receptors are already being engaged by their natural ligands (growth factors, hormones, or matrix proteins), that are abundant in serum and typically present in the micromolar range. Therefore, the targeting peptide must compete with these abundant native ligands for the sparse available free surface epitopes. A significant peptide affinity advantage is needed to compete with naturally occurring multivalent ligands, which may not bind the same epitope. Targeting may be impossible or ineffective for heavily serum-loaded receptors like the transferrin receptor or integrins. Fast dynamic exchange of bound peptide will decrease the intracellular delivery efficacy by reducing residence time on the cell surface and payload accumulation. The use of peptides in patients with elevated serum levels of the ligand (due to pathology or other physiological factors) is likely to result in low delivery and inter-subject variability. Another major challenge for saturable receptors is that increasing the dose of siRNA only results in limited or no improvement of intracellular uptake efficiency due to the saturated number of surface epitopes. Excessive peptide dose may saturate the clearance receptors in the liver and kidney and result in a longer half-life and unwanted side effects. All of these saturation effects are non-linear, making it difficult to determine the effect of dose increases during efficacy studies and to know when an optimum dose is reached. Therefore, researchers have tried to avoid receptor saturation by splitting up the peptide doses and injecting it over a prolonged period. Alternatively, they attempt to transiently increase the expression of the receptor target by injecting the peptide on an empty receptor. Both approaches increase the complexity of the treatment and may cause unintended biological response loops.
In this sense, the design of peptide-siRNA conjugates to some extent is a balancing act. On one hand, high-affinity and selective receptor binding is critical for targeting the tumor tissue, on the other hand, strong association often hinders the escape of the payload from endosomes. Moreover, cationic charges may condense the siRNA into nanoparticles, but will also lead to non-specific uptake. An increase in peptide density can result in more robust complexes but might also enhance peptide-associated toxicity. Given that each parameter affects the other parameters to some extent, it is virtually impossible to optimize one parameter without impairing another. Successful systems thus often aim at a compromise in which sub-maximal efficiencies in individual steps are exchanged for a global therapeutic window in which tumor accumulation, endosomal escape and cytosolic release are both sufficiently high. In the following, the antagonistic parameter pairs mentioned above are detailed and some practical compromises between them are discussed that allow to maintain a certain therapeutic index without introducing unnecessary molecular complexity for reasons of scalable synthesis.
Fig. 2 Rational design strategies to enhance peptide targeting efficacy. 2,5
Peptides with high affinity for the target tumor antigens are needed to rapidly saturate them and to quickly accumulate in the tumor. However, a high-affinity peptide can form very stable complexes with siRNA. Intracellular unpacking of siRNA would require dissociation of the complex, and highly stable complexes tend to persist without releasing the cargo. On the other hand, if binding is very weak, the siRNA may be released in the bloodstream before reaching the target cell. Therefore, it is necessary to design peptides with intermediate affinity (too weak to compete with other potential binding partners of the receptor, and too strong to prevent intracellular unpacking). This can be done by adding acid-labile linkers, or by mixing high affinity domains with low-affinity spacers, creating a mixed peptide that "lets go" after internalization without compromising targeting affinity. Addition of endosomolytic motifs (histidine-rich, fusogenic peptides) to the targeting domain can facilitate membrane disruption. However, this can also disrupt the conformation needed for binding to the receptor. Stabilizing the targeting domain through cyclization or hydrocarbon stapling, while leaving the lytic domain flexible can solve this issue, allowing two separate functions in one peptide backbone. Another strategy is to use a cleavable linker, which separates the two activities into two arms. In this case, the targeting arm remains bound to the receptor, while the lytic arm is released after endosomal proteolysis.
Strong cationic charges are required to condense the anionic siRNA into nanometric complexes. However, too strong a positive charge causes aggregation and fast opsonization by serum proteins. The surface charge of the complex can be adjusted by mixing cationic residues with neutral or anionic amino acids in order to obtain an overall positive charge with low zeta potential. In this way, complexes can be stabilized without promoting non-specific binding. Histidine is also often used as a pH-titratable group that is cationic at physiological pH (required for complexation) but protonated in the acidic environment of endosomes (required for membrane disruption). As a result, endosomal escape is ensured without compromising stability in the blood. However, such strong positive charges also cause adherence to anionic basement membranes in lung and kidney, resulting in accumulation that hides tumor targeting. Charge can also be shielded by hydrophobic residues (phenylalanine, tryptophan) by intramolecular stacking, which reduces the overall polarity of the peptide but keeps the condensation power. Shielding the charge through PEGylation or by addition of short anionic peptides also reduces non-specific uptake. They must however be added distal to the binding epitope to avoid steric inhibition of receptor binding. The balance is often found through iterative charge-screening libraries instead of single point mutations.
The improved potency of larger siRNA duplexes (27-mer Dicer substrates) comes at the expense of using more peptide chains for condensation and more extensive changes to the molecular weight (which also affects biodistribution properties due to elimination via renal filtration). Shorter 19-mer duplexes are easier to complex but may result in inadequate RNAi activity if the endosomal escape efficiency is low. Duplex length is thus typically chosen based on the condensation capacity of the peptide: small peptides (<15 mer) typically use 19-mer siRNA, while larger peptides or dendritic scaffolds are matched with 27-mer Dicer substrates without exceeding a 5 nm hydrodynamic radius. It is also critical that the complexes are mononucleic (carrying only a single siRNA molecule) and are not aggregates with multiple siRNA molecules, as this can lead to dosing challenges. Peptide-to-siRNA ratios on the order of 20:1 are often used to improve stability, but at the cost of increased risks of membrane disruption in healthy tissues. Such toxicity can be limited through use of cleavable linkers to decrease peptide density post-internalization, or by using peptides whose lytic activity is pH gated so that peptide activity is limited to the acidic environment of the endosome. Alternatively, the peptide can be made conditional (e.g. unfolds into its lytic conformation only after binding to its target receptor) so that the membrane-disruptive activity is only available inside the cell.
In general, the therapeutic potential of peptide-siRNA conjugates is maximized if the micro-environment of the disease has a well-defined, endocytic receptor that is not expressed (or expressed at negligible levels) in vital organs. In such a case, the small size of the peptide and the fast pharmacokinetics are assets, allowing the construct to saturate the receptor before renal clearance. When the target is more widespread, the receptor is masked by stromal deposits, or when non-specific uptake is stronger than the binding, peptide conjugates will probably not be the most efficient delivery system and larger, less specific carriers will perform better. The balance, therefore, is not one of absolute affinity for the peptide, but rather of receptor biology: ratio, accessibility and internalization rate need to be both high for peptide-based delivery to outcompete other approaches.
Optimal targets undergo ligand-mediated endocytosis within minutes of binding and co-internalize the siRNA cargo to early endosomes, which mature into late compartments from which escape occurs. The best candidates are growth-factor receptors, integrins, and nutrient transporters that cluster upon binding and traffic along well-studied endocytic pathways. Peptides can often be designed to bind to these receptors in a ligand-independent manner without inducing downstream signaling, and are taken up without disturbing the biology of the cell. The rate of internalization is crucial; if the target is poorly internalized and tends to recycle slowly to the cell surface, it will limit the amount of cargo that reaches the cytosol and renders high-affinity binding moot. Designers screen for receptor trafficking kinetics early in the process and drop targets that are unable to deliver the cargo to a point beyond the plasma membrane within 30-60 minutes. The key to success is a quantitative expression gap, often an order of magnitude or more, between diseased tissue and the organ that normally expresses the highest levels. This biological filter effectively overcomes a moderate binding affinity for the targeting peptide. It can be measured through transcriptomic atlases and quantitative immunohistochemistry; qualitative staining is not sufficient. The best candidates are those whose expression in healthy tissue is well below the threshold of PET imaging, so even picomolar off-target binding will not result in any signal.
When the target receptor is present on liver sinusoids, pulmonary capillaries or renal glomeruli where a high flow per unit mass is a normal physiological condition, charge-driven or hydrophobic adhesion dominates specific binding. In such instances, even if a peptide binds with high affinity, it saturates with healthy tissue on account of simple surface area and flow, diminishing selectivity. Larger carriers, like antibody-siRNA conjugates that recycle via FcRn or PEGylated nanoparticles, can discriminate better since their size limits access to normal tissue vasculature. Peptide approaches reach a point of cost-ineffectiveness when the ratio of specific to non-specific uptake drops below two-fold. After this point, any attempt at performance enhancement via engineering of antibody fragments, shielding agents or cell-penetrating peptides is likely futile. If a target receptor is buried in a desmoplastic stroma, trapped in tight junctions or found on a hypoxic core with diminished perfusion, it is not available to peptides in circulation. Furthermore, if the receptor is an inherently slow recycler or undergoes trafficking to degradative lysosomes rather than late endosomes accessible to the cytosol, the siRNA payload is destroyed en route. In this case, protease activated pro-drugs or small-molecule-siRNA conjugates that passively diffuse into the interstitium before binding outperform peptide active targeting approaches. Peptides are only considered as targeting elements for receptors whose extracellular domains sufficiently protrude beyond basement membranes, are accessible to targeting ligands within the confines of a single capillary transit and can therefore translate synthetic effort into measurable gene silencing.
Effective siRNA delivery depends on more than achieving cellular uptake. In many programs, functional gene silencing is limited by instability, non-specific distribution, or failure to reach the cytosol after internalization. Our targeting peptide services are designed to address these challenges by integrating siRNA compatibility, targeting precision, and delivery feasibility into a unified development framework. Rather than treating peptides as generic carriers, we engineer targeting peptides specifically for the biological and physicochemical requirements of siRNA payloads.
Payload-Aware Sequence Engineering: siRNA imposes unique constraints on peptide design due to its size, charge density, and sensitivity to degradation. Our payload-aware sequence engineering approach evaluates how peptide length, charge distribution, and secondary structure influence siRNA association, stability, and targeting behavior. Peptide sequences are optimized to form stable peptide-siRNA constructs without excessive positive charge that would otherwise drive non-specific uptake. This balance helps preserve tissue and cell-type selectivity while maintaining sufficient association strength for delivery.
Stability and Selectivity Optimization: Stability and selectivity are tightly linked in peptide-mediated siRNA delivery. We optimize peptide designs to resist serum degradation and premature dissociation while actively minimizing non-target interactions. This includes sequence-level modifications to improve protease resistance and fine-tuning of affinity to ensure receptor-mediated uptake dominates over passive electrostatic interactions. The goal is to achieve functional siRNA delivery driven by targeting rather than by non-specific accumulation.
Target Suitability Evaluation: Not all receptors that bind targeting peptides are suitable for siRNA delivery. Our feasibility assessment includes a target suitability evaluation that examines receptor expression patterns, internalization pathways, and compatibility with endosomal escape requirements. Targets that primarily support surface binding or slow recycling are flagged early, as they are less likely to enable efficient cytosolic siRNA release despite apparent uptake.
Early Risk Identification: Many failure modes in siRNA delivery can be identified early, including receptor saturation, competition with endogenous ligands, and loss of selectivity at higher doses. We focus on early risk identification to determine whether peptide-mediated delivery is likely to provide a meaningful advantage over alternative approaches. This risk-aware assessment helps teams avoid advancing peptide designs that improve uptake metrics without delivering functional gene silencing.
If your siRNA program achieves uptake but delivers inconsistent or limited gene knockdown, an early technical discussion can help clarify whether peptide design, target selection, or delivery architecture is the underlying constraint. Discuss your siRNA delivery challenge with our scientists to evaluate feasibility, identify key risks, and define a peptide strategy optimized for functional and selective siRNA delivery.
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