A failed CPP-oligonucleotide conjugate rarely fails for only one reason. Low conjugation yield may originate from an unstable functional group, but the same project may also suffer from purification loss, sequence-driven aggregation, poor endosomal escape, or an assay that measures uptake without confirming functional delivery. For peptide chemists, oligonucleotide scientists, and delivery researchers, the most productive response is not to repeat the same synthesis with more starting material. It is to diagnose where the failure entered the workflow and decide whether the next attempt requires a chemistry change, a sequence redesign, a purification adjustment, or a stronger biological control set.
CPP-oligonucleotide conjugates combine two chemically and biophysically different molecules. The cell-penetrating peptide may be cationic, amphipathic, hydrophobic, oxidation-sensitive, or prone to self-association. The oligonucleotide may carry a dense negative charge, bulky modifications, terminal protecting groups, phosphorothioate linkages, steric constraints, or hybridization requirements. A linker that is efficient during conjugation may be too stable for intracellular release, while a releasable linker may be sensitive during storage or purification. As a result, troubleshooting must connect analytical data with formulation behavior and cell assay results.
The first step is to define the failure mode precisely. "It did not work" can mean low conversion in the conjugation reaction, a broad analytical profile, poor recovery after purification, precipitation during formulation, weak cellular association, strong uptake but no gene modulation, or unacceptable cytotoxicity. Each problem points to a different decision path. A project that shows a clean conjugate peak but weak activity should not be redesigned in the same way as a project that never produced the expected conjugate mass.
Low yield usually means that the reactive peptide and oligonucleotide ends did not meet under suitable chemical conditions, or that one component degraded before the reaction could proceed. Common causes include hydrolysis of activated esters, thiol oxidation, incomplete deprotection, poor solubility of the CPP, steric hindrance at the oligonucleotide terminus, incompatible buffer additives, or pH conditions that reduce nucleophilicity. Before changing the entire construct, review the actual reaction evidence: starting material purity, functional group confirmation, reaction time course, mass balance, and whether unreacted peptide or unreacted oligonucleotide remains dominant.
Multiple peaks can arise from incomplete conjugation, side reactions, oxidation products, deletion peptides, oligonucleotide impurities, linker isomers, over-conjugation, salt adducts, or conformational/aggregation states that separate during chromatography. A single LC trace is rarely enough to assign the cause. Pair chromatographic data with mass spectrometry, oligonucleotide-specific detection, peptide-specific detection, and, when needed, denaturing gel or capillary electrophoresis. If several peaks have similar mass but different retention times, conformational or counterion effects may be involved. If the masses differ by predictable increments, linker or protecting-group chemistry may be responsible.
Solubility failure often appears after conjugation rather than during peptide or oligonucleotide analysis alone. A highly cationic CPP and an anionic oligonucleotide can form intramolecular or intermolecular ion-pairing structures. Amphipathic or hydrophobic CPPs can promote self-association. Phosphorothioate oligonucleotides may further alter binding to proteins and surfaces. Precipitation may also be triggered by desalting, lyophilization, organic solvent removal, divalent cations, high ionic strength, acidic pH, or concentration above the construct's practical solubility limit.
Weak uptake may indicate that the CPP is poorly matched to the cell type, the oligonucleotide cargo is too bulky or too highly charged for the selected design, the conjugate is aggregated and unavailable, or the assay underestimates cell-associated material. Some constructs bind strongly to serum proteins or plasticware, reducing effective exposure. Others require serum-free conditions to show uptake but lose performance in more biologically relevant media. Uptake should be measured with controls that distinguish surface binding from internalization and intact conjugate from degraded labeled fragments.
This is one of the most common and misleading outcomes in CPP delivery studies. A fluorescent signal may show cellular association, but the oligonucleotide may remain trapped in endosomes, may not release from the CPP, may be attached at a site that interferes with hybridization, or may degrade before reaching the active compartment. For antisense, splice-switching, siRNA, miRNA inhibitor, or aptamer projects, functional activity depends on intracellular localization, accessibility, target engagement, and assay timing—not uptake alone.
Cytotoxicity can result from the CPP sequence, high local positive charge, membrane perturbation, impurities, residual solvents, endotoxin or nuclease contamination, formulation excipients, fluorescent labels, or excessive dosing. Toxicity can also mask real biological activity by reducing viable cell number, changing gene expression nonspecifically, or activating stress pathways. A useful toxicity investigation includes free CPP, free oligonucleotide, unconjugated mixture, linker-only control when feasible, scrambled sequence control, and dose-response testing over the same exposure window used for functional readout.
| Problem | Possible Cause | Diagnostic Clue | Corrective Strategy | Service Support |
| Low conjugation yield | Unstable functional group, oxidized thiol, steric hindrance, incompatible pH | High remaining starting material; weak or absent expected mass | Confirm functional groups, adjust buffer, change linker, optimize stoichiometry | Reaction design review and conjugation condition optimization |
| Multiple product peaks | Side products, deletion sequences, oxidation, partial conjugation, aggregation | Several HPLC peaks with related or ambiguous masses | Use orthogonal analytics, revise purification method, improve starting material purity | Analytical interpretation and purification strategy support |
| Poor solubility | Hydrophobic CPP, charge complexation, counterion effects, high concentration | Turbidity, precipitation after desalting, broad chromatographic behavior | Add spacer, alter CPP sequence, screen pH/salt, use gentler concentration conditions | Sequence and formulation troubleshooting |
| Weak uptake | CPP not suited to cell type, aggregation, serum binding, poor exposure | Low fluorescence or weak intracellular signal after washing | Compare CPP variants, add labeled analog, evaluate serum and dose conditions | Design of matched labeled and unlabeled control constructs |
| Uptake but weak activity | Endosomal trapping, linker not releasing, attachment site blocks hybridization | Strong cell signal but minimal target modulation | Test release linker, move attachment site, add functional positive control | Linker and attachment-site redesign |
| Cytotoxicity | Excess cationic charge, membrane disruption, impurity, label or formulation effect | Reduced viability at active or subactive concentration | Lower dose, modify CPP, improve purity, separate toxicity from target effect | Control peptide and purity specification planning |
Table 1 Troubleshooting Matrix for CPP-Oligonucleotide Conjugates
Yield troubleshooting should begin with the simplest question: was each starting material chemically competent at the time of reaction? A CPP-oligonucleotide conjugation can fail even when both components look acceptable by routine purity analysis. The peptide may contain the correct main sequence but an oxidized cysteine. The oligonucleotide may contain the correct base sequence but a partially hydrolyzed or sterically masked terminal modifier. The linker may be theoretically appropriate but unstable under the actual handling conditions. A structured review prevents unnecessary redesign.
Many conjugation strategies depend on reactive handles that are sensitive to water, oxygen, reducing agents, nucleophiles, or time. Activated esters can hydrolyze before reacting with amines. Thiols can oxidize to disulfides or react with unintended electrophiles. Maleimide groups can undergo hydrolysis or exchange reactions depending on conditions. Azide-alkyne chemistries may be affected by reagent quality, catalyst compatibility, or oligonucleotide sensitivity. When yield is low, confirm the active functional group immediately before conjugation rather than relying only on the supplier's original certificate of analysis.
Practical diagnostics include comparing fresh and stored material, running a small-scale functional group test, checking mass shifts consistent with oxidation or hydrolysis, and reviewing freeze-thaw history. If the reactive group is unstable, a more stable precursor strategy may be needed. In some projects, it is better to generate the reactive handle immediately before conjugation or to switch to a linker that tolerates aqueous conditions and the required oligonucleotide chemistry.
CPP sequences containing cysteine, methionine, tryptophan, or multiple basic residues may show oxidation, deletion impurities, or side reactions that complicate conjugation. A cysteine intended for thiol-maleimide conjugation must remain reduced and accessible. Methionine oxidation may change hydrophobicity and retention behavior. Strongly cationic peptides can bind to oligonucleotides noncovalently, creating apparent conversion problems or broad peaks even before covalent reaction is complete.
Review peptide LC-MS data carefully, not only the reported purity percentage. A peptide preparation that is 85–90% pure may still be problematic if the major impurity contains the same reactive residue or co-elutes with the desired product. For sensitive sequences, consider using a protecting strategy, a terminal spacer, gentler storage, or a conjugation site placed away from oxidation-prone or aggregation-prone motifs.
Oligonucleotide terminus design can strongly affect conjugation. A bulky 5′ or 3′ modification, structured sequence, high secondary structure, phosphorothioate content, or nearby locked nucleic acid modification may reduce accessibility of the reactive handle. Even when the handle is present, it may be physically shielded by the oligonucleotide's conformation or by ion-pairing with the CPP.
If steric hindrance is suspected, compare conjugation to a short model oligonucleotide with the same terminal chemistry, evaluate the effect of temperature within stability limits, and consider adding a spacer between the oligonucleotide and reactive group. A short hydrophilic spacer can improve access without substantially changing the biological design. For hybridization-dependent applications, the spacer may also reduce interference between the CPP and the recognition region.
Buffer choice can make an otherwise sound conjugation fail. Primary amine buffers can compete with amine-reactive chemistry. Reducing agents can interfere with thiol-reactive systems. Chelators, salts, detergents, and organic co-solvents may improve solubility but inhibit the reaction or complicate purification. pH controls the charge state and nucleophilicity of reactive groups, while also affecting oligonucleotide stability and CPP solubility.
A useful troubleshooting experiment is a small reaction matrix rather than a single repeated reaction. Screen a narrow pH range, peptide-to-oligonucleotide ratio, reaction concentration, co-solvent percentage, and reaction time while monitoring conversion by an analytical method that can distinguish free oligonucleotide, free peptide, and conjugate. The goal is not simply to maximize peak area; it is to identify conditions that produce a clean, recoverable product.
Purification is often where an apparently successful reaction becomes a low-recovery project. CPP-oligonucleotide conjugates can behave differently from either starting material. They may bind strongly to reversed-phase media, elute broadly, form ion-pair complexes, stick to filters, precipitate during solvent removal, or lose material during desalting. A purification method that works for an unmodified oligonucleotide may not work for a conjugate containing an amphipathic CPP.
Co-elution is common when the CPP changes the oligonucleotide's hydrophobicity only modestly or when the peptide and conjugate both interact strongly with the chromatographic system. UV detection at a single wavelength may mislead interpretation because oligonucleotides and peptides absorb differently. Monitoring both nucleic-acid and peptide-relevant wavelengths can improve peak assignment. Mass spectrometry, denaturing PAGE, capillary electrophoresis, or ion-exchange methods may be needed to confirm identity and purity.
Corrective options include changing gradient slope, ion-pairing conditions, column chemistry, temperature, loading amount, or purification mode. In some cases, anion-exchange chromatography better separates oligonucleotide-containing species, while reversed-phase HPLC better resolves hydrophobic peptide-related impurities. The best method depends on whether the main impurity is unconjugated oligonucleotide, unconjugated peptide, truncated oligonucleotide, oxidized peptide, or a closely related conjugate side product.
Aggregation during HPLC may appear as broad peaks, poor reproducibility between injections, pressure changes, low recovery, or product appearing in multiple fractions. Organic solvent, ion-pairing reagents, pH, concentration, and temperature can all shift the balance between monomeric and aggregated states. Highly cationic CPPs may interact with anionic oligonucleotide regions within or between molecules, while hydrophobic CPP motifs may promote self-association under gradient conditions.
Troubleshooting should compare analytical-scale and preparative-scale behavior. A conjugate that looks acceptable at low loading may aggregate at higher concentration. Reducing injection concentration, changing counterions, adding a hydrophilic spacer, using gentler gradient conditions, or switching to a purification method with less hydrophobic stress can improve recovery. Avoid assuming that a broad peak always means chemical impurity; it may also reflect reversible association.
Desalting and buffer exchange can cause major losses through membrane adsorption, precipitation, incomplete elution, or concentration above the solubility limit. CPP-containing conjugates may bind to plastic, glass, filtration membranes, or desalting resin. Loss may also occur when ion-pairing reagent is removed too quickly, changing the conjugate's solvation environment.
To identify the loss point, measure material before and after each handling step rather than only at final lyophilization. Use low-binding consumables, evaluate recovery from the device using a small test sample, avoid unnecessary drying cycles, and consider whether the final salt form is compatible with solubility and biological testing. For very difficult constructs, formulation screening may need to be integrated with purification rather than treated as a separate final step.
Solubility is a design property, not just a formulation detail. CPP-oligonucleotide conjugates carry competing features: the oligonucleotide is generally hydrophilic and polyanionic, while the peptide may be cationic, hydrophobic, amphipathic, or conformationally flexible. The final construct may behave as a compact ion-paired species, an extended monomer, a reversible aggregate, or an insoluble complex depending on sequence and conditions.
Hydrophobic or amphipathic CPPs can improve membrane interaction but may reduce aqueous handling. Sequences containing long hydrophobic stretches, aromatic residues, or strong amphipathic helices can self-associate, especially after attachment to an oligonucleotide that changes the overall shape and local charge distribution. Aggregation risk may increase during concentration, lyophilization, or buffer exchange.
Corrective strategies include adding a hydrophilic spacer, reducing hydrophobic residue density, moving the attachment site, testing a less hydrophobic CPP analog, or using a formulation that maintains the conjugate below its aggregation threshold. However, reducing hydrophobicity may also reduce membrane interaction, so redesign should be guided by both analytical behavior and cell assay performance.
Charge balance is central to CPP-oligonucleotide behavior. Highly arginine- or lysine-rich CPPs can associate strongly with the oligonucleotide's phosphate backbone. This can be useful for cellular association but harmful if it creates compact aggregates, masks the oligonucleotide's recognition sequence, prevents release, or increases nonspecific membrane disruption. Conversely, reducing cationic charge too much may weaken uptake.
A practical approach is to compare charge variants rather than make a single large redesign. Shortening the CPP, substituting selected residues, adding neutral hydrophilic spacers, or changing the attachment terminus can alter charge presentation without eliminating the delivery function. For biological studies, include controls that distinguish sequence-specific oligonucleotide activity from nonspecific cationic peptide effects.
Spacers can reduce steric crowding, improve solubility, and separate the CPP from the oligonucleotide recognition region. Short alkyl spacers may provide distance but not enough hydrophilicity. PEG-like spacers can improve hydration and flexibility, but excessive spacer length may alter uptake, intracellular trafficking, or release kinetics. Amino acid spacers such as glycine-serine motifs may be useful when peptide synthesis compatibility and flexibility are priorities.
Spacer selection should be based on the suspected failure mode. If conjugation yield is low because the reactive handle is inaccessible, a short spacer near the oligonucleotide may help. If aggregation is driven by peptide-oligonucleotide ion pairing, a hydrophilic spacer between the domains may help. If biological activity is weak because the attachment site interferes with hybridization, moving the linker or increasing distance from the target-binding region may be more important than changing spacer chemistry alone.
Counterions can influence solubility, chromatographic behavior, lyophilization, and cell assay compatibility. Material isolated from ion-pairing reversed-phase HPLC may require careful handling before biological use. Salt exchange, buffer selection, pH, concentration, and freeze-thaw conditions can all change whether the conjugate remains soluble. Divalent cations may also promote unwanted association in some systems.
Formulation troubleshooting should be empirical but controlled. Test a small panel of pH and ionic strength conditions, monitor clarity and recovery, and use analytical confirmation after storage. Avoid evaluating biological activity from a formulation that contains visible precipitate or unknown aggregate content, because apparent low activity or high toxicity may reflect physical instability rather than the intended design.
Biological troubleshooting should begin only after the construct's identity, purity, and formulation state are reasonably understood. Otherwise, the cell assay becomes a black box. Poor activity may reflect insufficient exposure, weak uptake, endosomal trapping, lack of release, wrong attachment site, degradation, inaccessible target sequence, inappropriate assay timing, or poor control design. The strongest troubleshooting studies separate delivery, release, target engagement, and toxicity into measurable steps.
CPPs often improve cell association and internalization, but internalization does not guarantee cytosolic or nuclear availability. A conjugate may accumulate in endosomal or lysosomal compartments and still show strong fluorescence microscopy signal. For antisense or RNA-targeting applications, the active oligonucleotide must reach the compartment where its target and mechanism are accessible. Endosomal escape can therefore be the limiting step even when uptake appears successful.
Useful diagnostics include microscopy with endosomal markers, functional positive controls, time-course studies, comparison with a known transfection method, and assays that measure target modulation rather than fluorescence alone. When endosomal escape is suspected, redesign may involve CPP sequence selection, linker release strategy, formulation changes, or use of delivery-enhancing conditions appropriate to the research model.
Linker chemistry must match the intended mechanism. A stable linker may be ideal when the CPP should remain attached, but it may reduce activity if the oligonucleotide must separate from the peptide to hybridize, recruit proteins, or avoid steric blocking. A cleavable linker may improve intracellular availability but can reduce stability during purification, storage, or extracellular exposure. Disulfide, acid-labile, enzyme-sensitive, and noncleavable linkers each carry different trade-offs.
If uptake is adequate but activity is poor, compare a stable-linker construct with a releasable-linker construct when scientifically appropriate. Also test whether the free oligonucleotide is active under a validated delivery condition. If the free oligonucleotide lacks activity even with a positive transfection control, the problem is not primarily the CPP conjugation design.
The position of CPP attachment can influence oligonucleotide hybridization, protein recruitment, nuclease resistance, and intracellular trafficking. A 5′ attachment may be acceptable for one antisense design but disruptive for another. A 3′ attachment may protect against exonuclease activity in some contexts but interfere with other mechanisms. Internal attachment may provide spatial advantages but increases synthesis and analytical complexity.
Attachment-site troubleshooting should consider the oligonucleotide's mechanism of action. For RNase H-dependent antisense designs, steric effects and gapmer architecture matter. For splice-switching oligonucleotides, target accessibility and nuclear delivery are critical. For siRNA-related designs, guide strand loading and duplex architecture must not be disrupted. Moving the CPP away from the functional region can sometimes restore activity without changing the CPP itself.
Without controls, it is difficult to know whether the conjugate failed, the assay failed, or the biological hypothesis was incorrect. At minimum, consider free oligonucleotide, free CPP, unconjugated CPP plus oligonucleotide mixture, scrambled or mismatch oligonucleotide conjugate, positive delivery control, untreated cells, vehicle control, and viability readout. For uptake studies, include labeled CPP alone and labeled oligonucleotide alone when feasible. For functional studies, include a validated assay endpoint and an exposure time suitable for the mechanism.
Controls also help detect artifacts. A fluorescent label can change uptake. A CPP can produce nonspecific gene expression changes. A high dose can reduce apparent target expression by reducing viable cell number. A positive microscopy signal may represent surface-bound material if the wash and quenching strategy is inadequate. Good controls turn a failed experiment into a redesign map.
Before reordering, consolidate the evidence into a redesign brief. The brief should include the peptide sequence, oligonucleotide sequence and chemistry, attachment site, linker structure, functional groups, reaction conditions, crude analytical data, purification method, final purity, formulation, storage conditions, cell type, dose, exposure time, uptake method, functional assay, and control outcomes. A redesign decision made from complete information is more likely to solve the true failure mode.
CPP sequence modification is appropriate when aggregation, toxicity, poor uptake, or endosomal trapping appears sequence-driven. Options include reducing hydrophobicity, adjusting arginine/lysine density, shortening the peptide, introducing hydrophilic residues, changing chirality or terminal modification for research comparison, or testing an alternative CPP family. The goal is not always maximum uptake; it is useful intracellular delivery with acceptable solubility and cell tolerance.
Linker redesign is appropriate when conjugation yield, stability, release, or steric accessibility is the likely bottleneck. A different linker may improve aqueous reaction efficiency, reduce side products, separate the CPP from the oligonucleotide, or enable intracellular release. Linker changes should be evaluated against the full workflow. A linker that improves biological activity but collapses during purification may not be practical without additional process changes.
Attachment-site adjustment is often underestimated. Moving the CPP from 5′ to 3′, from 3′ to 5′, or away from a critical recognition region can improve hybridization, reduce steric blocking, or change intracellular behavior. For modified oligonucleotides, attachment must be compatible with the existing chemical architecture. When possible, compare two attachment-site variants rather than assuming one terminus is universally superior.
If biological interpretation is unclear, adding controls may be more valuable than changing chemistry immediately. A labeled analog can show uptake and localization, but it should be used cautiously because labels can alter behavior. Matched unlabeled and labeled constructs help separate imaging convenience from functional performance. Scrambled conjugates, mismatch controls, and free component controls help distinguish delivery failure from sequence-independent effects.
Early feasibility studies may tolerate moderate purity for screening, but troubleshooting often requires stronger analytical definition. Specify the desired purity threshold, identity confirmation method, residual unconjugated oligonucleotide limits when important, residual peptide considerations, salt form, endotoxin requirements if relevant to the cell system, and storage recommendations. For difficult constructs, request analytical traces and mass data that allow the team to evaluate whether the material is suitable for the intended assay.
| Design Change | Intended Benefit | Technical Trade-Off | When to Use |
| Reduce CPP hydrophobicity | Improve solubility and reduce aggregation | May reduce membrane interaction or uptake | Visible precipitation, broad HPLC peaks, poor recovery |
| Adjust cationic residue density | Balance uptake, toxicity, and oligonucleotide binding | May alter intracellular trafficking and potency | High toxicity, nonspecific effects, or strong aggregation |
| Add hydrophilic spacer | Reduce steric hindrance and improve hydration | May change distance, flexibility, and uptake profile | Low conjugation yield, poor hybridization, aggregation |
| Switch to cleavable linker | Improve intracellular oligonucleotide availability | May reduce extracellular or purification stability | Good uptake but weak functional activity |
| Use noncleavable linker | Improve construct stability and simplify analysis | May limit activity if release is required | Premature cleavage, unstable storage, ambiguous mass profile |
| Move attachment site | Reduce interference with hybridization or mechanism | Requires new oligonucleotide modifier and validation | Free oligo works, conjugate enters cells, activity remains weak |
| Add labeled analog | Track uptake and localization | Label may alter physicochemical behavior | Unclear delivery, trafficking, or assay exposure |
| Raise QC requirements | Improve interpretability and reproducibility | May reduce yield and increase production complexity | Multiple peaks, toxicity concerns, sensitive functional assays |
Table 2 Redesign Options Before Reordering
CPP-oligonucleotide troubleshooting benefits from coordinated review of peptide design, oligonucleotide chemistry, conjugation strategy, purification behavior, and biological assay requirements. Creative Peptides can support research-use projects by reviewing failed reaction details, evaluating peptide sequence liabilities, advising on functional group placement, comparing linker options, and developing practical conjugation and purification strategies for difficult constructs.
For projects with low yield, the review may focus on reactive handle stability, peptide oxidation risk, oligonucleotide modifier accessibility, reaction pH, buffer compatibility, and analytical confirmation. For projects with aggregation or poor recovery, the review may focus on CPP hydrophobicity, charge distribution, spacer design, salt form, purification mode, and concentration conditions. For projects with weak cellular activity, the review may focus on uptake versus functional delivery, linker release, attachment-site effects, and the design of matched control peptides or labeled analogs.
The most useful technical inquiry includes the peptide sequence, oligonucleotide sequence or chemistry description, intended attachment site, linker design, functional groups, reaction conditions, crude and purified analytical data, purification method, formulation or storage conditions, intended cell model, uptake assay, functional assay, and any observed toxicity. These details allow the technical team to distinguish a chemistry problem from a purification problem, a formulation problem from a trafficking problem, and an assay problem from a true design limitation.
If your CPP-oligonucleotide conjugate showed low conjugation efficiency, multiple product peaks, aggregation, weak uptake, uptake without activity, or unexpected cytotoxicity, share the failed reaction details and available analytical data for technical review. A structured redesign can help determine whether the next construct should change the CPP sequence, linker chemistry, attachment site, purification approach, control set, or QC requirements before additional synthesis resources are committed.
Low yield may result from unstable reactive groups, thiol oxidation, hydrolyzed activated esters, steric hindrance at the oligonucleotide terminus, incompatible buffer components, poor CPP solubility, or an unsuitable pH. Review starting material identity, functional group integrity, reaction time course, and crude LC-MS or related analytical data before redesigning the construct.
Aggregation often comes from hydrophobic CPP motifs, excessive cationic charge, intramolecular or intermolecular ion pairing with the oligonucleotide backbone, counterion effects, high concentration, or formulation changes during desalting and lyophilization. Hydrophilic spacers, CPP sequence modification, concentration control, and formulation screening may help.
Uptake does not guarantee functional delivery. The conjugate may remain trapped in endosomes, fail to release the oligonucleotide, attach at a site that blocks hybridization, degrade intracellularly, or reach the wrong compartment. Functional controls and localization studies are needed to separate uptake from target engagement.
Identify where loss occurs by measuring material after each purification, desalting, concentration, and drying step. Consider alternative chromatography modes, lower loading concentration, low-binding consumables, gentler gradient conditions, compatible counterions, and formulation screening before final lyophilization.
Useful controls include free oligonucleotide, free CPP, unconjugated CPP plus oligonucleotide mixture, scrambled or mismatch conjugate, positive delivery control, untreated and vehicle controls, viability readout, and matched labeled/unlabeled analogs when uptake or localization is being studied.
