Innovative Cell Penetration Peptide Technologies for Even, Young and Healthy Looking Skin

It is highly challenging to deliver the cosmetic actives and drugs to the skin because of the stratum corneum (SC). The permeability of large hydrophilic peptides or compounds across the stratum corneum in the outer skin surface is extremely low because of the lipophilic properties of the uppermost skin layer.

A short synthetic peptide was found to facilitate efficient transdermal protein drug delivery through the intact skin. Besides, the transdermal-enhancing activity of the peptide was sequence specific and dose-dependent. The peptide creates a temporary opening in the skin barrier to enable insulin or other drugs to reach systemic circulation. So peptides hold potential as drug delivery vehicles for the cosmetic purpose because of their simplicity, biocompatibility, and multi-functionality.

The delivery system was applied to treat melisma. Treatment with a cream formulation containing the transdermal peptide co-administered with other agents resulted in measurable inhibition of melanin production and melanocyte apoptosis. After four weeks application, patients demonstrated a significant lightening of facial hypermelanosis lesions and almost wholly restored dark lesions to normal skin color after 12 weeks. These innovative peptide technologies give the patients for even, young and healthy looking skin. Its topical application induces restoration of the skin’s most important structural dermal components.

LifeTein is working on a safe cell penetration peptide for the co-administration with another short cosmetic peptide. A few short peptide candidates have been proved to significantly and visibly improve the skin’s evenness and overall appearance, and reduce keratinocyte-induced activation of melanocytes. Together with the cell penetration peptides, the cosmetic peptide acts safely and as a result inhibits the skin pigmentation process, which is of major cosmetic concern.

Prolong the peptide bioactivity by reversible self-assembly

Self-assembly peptide into fibrillar nanostructures

Most of the potential therapeutic peptides have low solubility, chemical instability or low stability against protease. So it is essential to modify and optimize the peptides to improve the peptide bioavailability.

One novel approach is to create self-assembly, highly ordered and stable nanostructure. For instance, many peptide hormones including glucagon are stored in the form of β-sheet rich amyloid-like fibrils via a hydrogen bond network in the secretory cell.

The oxyntomodulin is a peptide with a potential to treat obesity and diabetes. It is a 37-amino-acid proglucagon-derived peptide hormone with sequence homology to both glucagon and glucagon-like peptide-1 (GLP-1). The oxyntomodulin peptide self-assembles into a stable nanofibril formulation and later on releases active peptide.

Here is the sequence of human oxyntomodulin: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala, HSQGTFTSDY SKYLDSRRAQ DFVQWLMNTK RNRNNIA.

Oxyntomodulin self-assembles into fibrillar nanostructures

  1. The peptide concentration was 10 mg/mL in water at an incubation pH of between 7.0 and 7.3 and low ionic strength (0.09% saline).
  2. The solution was incubated at 37 °C and agitated by orbital shaking.
  3. After incubation for five days, the solution turned turbid due to the formation of a suspension of aggregates. The conversion yield of the self-assembly of Oxyntomodulin into fibrillar nanostructures was estimated to be 99% under these conditions.
  4. The nanofibrils were next used to seed a solution of free peptide at 10 mg/mL in water. 5. The solution was incubated without agitation for one week and then diluted to 1 mg/mL in 0.09% saline for another 2–9 days of incubation at 37 °C and then nine days at room temperature.
  5. Free peptides were mainly in an α-helical conformation. The fibrillar Oxm showed the majority of β-sheet and some α-helical content.

Peptide nanofibrils dissociate to release intact peptide

  1. 1 mg/mL nanofibrils were incubated in water. The 37% of the peptide was released after four-hour incubation.
  2. In aqueous HCl, a 77% release was observed after only four hours. The peptide remained chemically intact after discharge from the nanofibrils


  1. The released peptide is active and nontoxic in vitro.
  2. The nanofibrils prolong peptide serum bioactivity in vivo. And there is no need to engineer or modify the original peptide. Other peptide hormones including glucagon, GLP-1, exendin-4, calcitonin, and gastric inhibitory peptide, are known to self-assemble. The self-assembly method may be used for the clinical application of reversibly self-assembling nanofibrils.

Controlling the bioactivity of a peptide hormone in vivo by reversible self-assembly. Nature Communications, volume 8, Article number: 1026 (2017)

Neoepitopes as cancer immunotherapy targets

Cancer is a patient-specific disease, where no two tumors are alike. The neoepitopes are very frequent in all cancers. Amino acid substitutions can yield neoantigens that are detected by the immune system. So the neoantigens have been used for the therapeutic purposes such as identifying cancer variant peptides for diagnosis and treatment.

The neopeptides have the following characteristics.

1. The 9-mer peptides are the most common among the high binding neoantigens.

2. The neopeptides have the hydropathy nature. The amino acid distributions, at all positions in neoepitopes of all lengths, contain more hydrophobic residues than the wild-type sequences.

3. Only a minority of predicted neoepitopes elicits protective tumor immunity. Peptide binding to a Human leukocyte antigen (HLA) molecule is a requirement for raising adaptive immunity.

How should we immunize against neoepitopes?

Since neoantigens are small peptides harboring tumor mutations, immunization with them usually needs strong immunostimulatory agents to produce an effective immune response.

Peptides as vaccines may not be able to stimulate the immune system powerfully enough on their own. Therefore, it is usually required to use an adjuvant in combination to elicit an effective immune response.

However, the MAPs-4 system, in which four copies of the same peptide epitope are synthesized on a lysine-based core, does not require a carrier protein, as the dense packing of multiple copies of an epitope in combination with a high-molar ratio produces a robust immunological response.

Accurate identification of neoepitopes and their subsequent use in cancer therapy is still in its nascent stages. With recent advances in Mass Spectrometry, faster and more precise identification of all expressed neoepitopes may be possible soon.

Neoepitopes as cancer immunotherapy targets: key challenges and opportunities

Protein-protein interactions for drug discovery

Protein-protein interactions (PPI) are highly specific electrostatic attractions between protein structures. The interactions regulate cell function and influence physiology and development. The mass spectrometry is often used to detect the protein-protein interactions. However, all proteomic screens are differential and require two samples such as IP and mock IP, POI and wildtype, or POI and knockout.

The peptide/ biotin-peptide or peptide/fluorescence-labeled peptide are excellent tools to study the protein-protein interactions. Check this link for details: Protein-Protein Interactions: Methods for Detection and Analysis.

The compounds that can modulate PPI are hard to discover because the proteins have multiple binding sites and the screening assays are not reliable. In many cases, two or more proteins may interact with one another and form a complex. The optical fluorescence-based methods such as the Cy5, Cy7, FAM, FITC, TAMRA-labeled peptides, or FRET assay are particularly useful in these circumstances. Click for more details:

The interactions between a fluorescently labeled or intrinsically fluorescent sample and a binding patterner are measured during the application. The changes in intrinsic fluorescence from tryptophan and tyrosine residues in the protein can be measured, which indicate transitions in the protein’s folding state.

The scientists have been working on fusion-based bifunctional proteins in cancer immunotherapy. The bifunctional protein sent an apoptotic signal to the tumor cells and enhanced their killing. The click chemistry is the perfect tool for the drug-protein or protein-protein conjugation. The more we understand the natural receptor-ligand complex and how it might signal, the better we can guide the design of therapeutic agonists. Click here for the peptide conjugation details:

Epitope mapping using synthetic peptides

Epitope mapping identifies antigen regions that serve as binding sites for antibodies. The overlapping linear peptides derived from the primary sequence of the antigen is frequently used for the epitope screening. Individual peptides can be divided into several fragments that overlap. The resulting overlapping peptide libraries can then be used for processes including continuous and linear epitope mapping.

For example, to map the epitope of an antibody, a few overlapping fragments spanning the target regions are constructed in an expression vector. These constructs are transiently transfected in cells and whole cell lysates collected after 48 hours are subjected to Western blotting with the antibody. To further map the region of this fragment, a series of overlapping peptides are synthesized. These peptides and lysates from the cells expressing target full-length gene or empty vector (negative) are performed by the dot blot.

Mapping epitopes quickly and accurately are challenging because the epitopes tend to be nonlinear on antigens. Combining binding specifically toward two distinct epitopes into a single molecule can significantly enhance the immunotherapeutic properties of monoclonal antibodies. Multivalent interactions are the most efficient at driving IgE receptor signaling pathways.

There are several useful tools for studying the antigen-antibody interactions.

  1. Use the MAPs as the immunogens. Multiple Antigenic Peptides (MAPs) are peptides that are branched artificially, in which Lys residues are used as the scaffolding core to support the formation of 8 branches with varying or the same peptide sequences. For example, one goal is to include different epitopes from different virus proteins in a single unit. The epitopes showing the right prediction of antigenicity and conserved in most serotypes of a virus are selected and assembled as the MAP.
  2. Screening combinatorial peptide libraries to optimize enzyme substrates and create high-affinity protein ligands. A critical biological application of custom peptide libraries is the characterization of the binding events that occur between specific proteins and their peptide ligands. A series of Overlapping Peptide, Truncation Peptide, Alanine Peptide Scanning, Scrambled Peptide, or Positional Peptide can be used for mapping and validating epitopes, the characterization of therapeutic antibodies, studying anti-antibody and neutralizing antibody actions in vitro.
Synthetic Peptide Library

Cell Penetration Peptide Synthesis

Tat, the transcription activator of the human immunodeficiency virus type 1 (HIV-1) viral genome, enters cells in a non-toxic and highly efficient manner. Tat is the first known cell-penetrating peptide.

CPPs have been used as a carrier to deliver proteins or genes into cells and tissues. In this study, a CPP library composed of 55 CPPs were used to deliver genes into plant cells. Many CPPs showed efficient penetration into plant cells. The Lys-containing CPPs have higher penetration efficiency in the plant than in animal cells. This could be due to differences in lipid composition and surface charge of the cell membranes. No correlations were detected between the penetration efficiency and the cationic, amphipathic, or hydrophobic properties of peptides.

D-R9 is composed of D-form amino acids. D-R9 bound preferentially to the membrane and did not penetrate the cytosol or vacuole. In mammalian cells, poly-Lysine-based CPPs are efficient and interact with membrane lipid head groups to induce wrapping of the membrane monolayers. Arg-rich peptides, such as the Tat peptide, are among the most efficient CPPs. Arg-rich CPPs may generate negative Gaussian membrane curvature to form pores or protrusions from endocytosis. The cell penetration efficiency of CPPs containing poly-Arg is higher than those containing poly-Lys. However, in a plant, Arg-rich CPPs are not the most efficient at penetrating plant cell.

CPP peptide property

CPP peptide property

How to do competition binding assay?

Endocrine fibroblast growth factors (FGFs) require Klotho transmembrane proteins as necessary co-receptors to activate FGF receptor (FGFR) signaling.

A series of peptides were synthesized by LifeTein and used for the competition binding assay. Both the KL1 and KL2 domains of β-Klotho participates in ligand interaction. The FGF19 peptide was used for alanine scanning mutagenesis. It was found that a single amino acid mutations in either region were sufficient to abolish β-Klotho binding. FGF19 and FGF21 function through β-Klotho to regulate glucose and lipid metabolism.

How to perform the solid-phase binding assay

1. The 96-well plates were coated overnight at 4 °C with 2 µg/mL of antibody in PBS.

2. Plates were washed twice with PBST and blocked with 3% (w/v) BSA in PBS for 1.5 hours at room temperature.

3. The conditioned media containing β-Klotho were added to the plates and incubated for 1.5 hours at room temperature.

4. Plates were washed a few times.

5. The peptide mutation FGF21 and an anti-β-Klotho antibody were biotinylated with EZ-Link Sulfo-NHS-LC-Biotin at the indicated concentrations.

6. After wash, streptavidin-HRP was used for detection.

7. EC50 values were determined.

How to do competition binding assay?

1. The WT and mutant peptides were custom synthesized and purified (>95% purity) by LifeTein.

2. Binding of FGF19 and FGF21 peptides to β-Klotho was assessed.

3. The β-Klotho ECD 6 × His, varying amounts of FGF19 and 21 peptides, and biotinylated human FGF19 or FGF21 protein were prepared.

4. The streptavidin donor beads and nickel chelate acceptor beads were added to the plates.

5.Plates were incubated for 3 hours at room temperature protected from light and read on the Plate Reader.

Reference: Scientific Reports, volume 8, Article number: 11045 (2018)




Tumor Targeting of Conjugated Synthetic Peptides

The folate receptor alpha (FRα) is highly expressed in ovarian cancer and not in normal tissues. An FRα binding peptide C7 (Met-His-Thr-Ala-Pro-Gly-Trp-Gly-Tyr-Arg-Leu-Ser, MHTAPGWGYRLS)  was found to bind to FRα expressing cells. This tumor-targeting peptide was proved by both phage homing experiment and fluorescence imaging.

Tumor Targeting of Conjugated Synthetic Peptides
1. The FITC conjugated peptide FITC-MHTAPGWGYRLS was dissolved in PBS.
2. The peptide was injected intravenously into a tumor-bearing nude mouse.
3. After 2 h, tumor and other organ tissues were harvested and analyzed using a fluorescence imaging system.

Cell internalization of Synthesized Peptide
1. Cells were seeded in 24 well plates containing coverslips and incubated for 24 hours in medium with FBS.
2. FITC conjugated peptide was incubated with the cells for 4 hours at 37 °C.
3. The cells were washed once with PBS and fixed in 4% paraformaldehyde.
4. Cells were washed three times with PBS and stained with DAPI for 20 min at room temperature.
5. Internalized fluorescent signals were imaged with a confocal microscope.

This tumor-specific peptide could be a potent and selective ligand for FRα. It has a great potential for delivery of cancer therapeutics or imaging agents to FRα expressing tumors.

Reference: Scientific Reports, volume 8, Article number: 8426 (2018)

tumor targeting peptides

tumor targeting peptides

Cell Penetration Peptide Targeting Cancer

Peptide Targeting Cancer

Cancer is expected to surpass the current number one, cardiovascular diseases by 2030 as the leading cause of death.

The targeting peptides can be considered as an alternative vehicle for the delivery of anti-cancer drugs because of their lower molecular weight and excellent tolerability by human bodies.

The following modification can prolong the half-life of the peptides from the degradation by blood proteases: forming cyclization within a peptide, blocking of the C- and N- terminus, replacement of standard L-type amino acids by their D-amino acid counterparts, using of unnatural amino acids incompatible with endogenous proteases.

About targeting peptides:
1. Somatostatin (SST) derivatives: Binding of the natural ligand somatostatin peptide to the receptors leads to inhibition of overexpressed SSTR2 and 5 in breast cancer. For example, cyclic SSTR agonist octreotide (fCfDWKTCT), selectively binds SSTR2 and 5.

2. Peptide-derivatives of gastrin-releasing peptide (GRP)

The gastrin-releasing peptide receptor is associated with the prostate and breast cancer. It was found that bombesin (YQRLGNQWAVGHLM) and its derivatives could be used as targeting peptide for detection of prostate cancer via PET and CT screening.

3. Peptides targeting tumor microenvironment

The glycoprotein prosaposin (PSAP) can inhibit metastases from breast and lung cancer in preclinical models. A cyclic PSAP peptide (DWLPK) could hinder metastatic spread and restrain tumor development. The synthetic antagonist of CXCR4 called NT21MP (LGASWHRPDKCCLGYQKRPLP) exhibits anti-tumor activities through decreased adhesion and migration of breast cancer cells. The peptide R (RACRFFC) targeting of CXCR4, showed capacities to remodel the tumor stroma.

4. Peptides targeting the tumor pH and temperature

The pHLIP (pH-Low Insertion Peptide), ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET, can insert into cell membranes as an a-helix under low pH conditions. The pHLIP peptide increases the uptake of the peptide-coated drug to tumors compared to the naked particles. Thus, the local tumor microenvironment can be used to trigger peptide drug formulations to respond accordingly.

The elastin-like polypeptides (ELP) is conjugated to the cell penetrating peptide Bac (RRIRPRPPRLPRPRPRPLPFPRPG) for improved cellular penetration to deliver gemcitabine for pancreatic cancer.

5. Peptides targeting tumor tissues

A widely used endothelial binding peptide is The tripeptide arginine-glycine-aspartic acid (RGD), an endothelial-binding peptide, has high specificity towards integrins for anti-tumor and anti-angiogenic treatments.

The radiolabeled, PEGylated RGD has been used as a PET-probe to detect gliomas, and the iron-oxide nanoparticles coupled RGD was used for the MR imaging of brain tumors.

The cyclic iRGD (CRGDKGPDC) is a prototypic tumor-penetrating peptide binding integrins. The iRGD peptide increased the tumor tissue penetration and the delivery of drugs, nanoparticles, or antibodies in vivo.

The cRGD (RGDdYK) and cilengitide (cRGDf [N-Me]V) with the combination therapy of temozolomide (TMZ) radiochemotherapy were used in the clinical trials.

A linear targeting peptide called CooP (CGLSGLGVA) binds the mammary-derived growth inhibitor (MDGI). The MDGI is a fatty acid binding protein that is highly expressed at the cell membrane of malignant glioma cells.

The coop peptide is a homing peptide targeting glioma cells and tumor-associated blood vessels. The chemotherapeutic drug conjugated CooP peptide can reduce the number of invasive tumor cells.

The cell penetration peptides are the cure for the difficult-to-access cancers such as brain tumors. This endothelial-specific peptide with enhanced penetrance would allow better passage of the drug conjugates through the blood-brain barrier.

The peptide drugs have the benefits of high specificity, low antigenicity, low cost and simple production. The peptides have the potentials for the development of therapy options for various tumors in the field of personalized medicine of cancer.



How to analyze the cell viability with peptides?

  1. Cells were seeded on 96-well culture plates (10000 cells/well) and incubated in 100 μL of DMEM containing 10% FBS.
  2. The medium was then replaced with fresh medium containing 10% FBS, and a peptide solution was added to each well at an appropriate concentration (for example 0.5uM, 1uM, 1.5uM, 2uM).
  3. After a 2-h incubation, Cell counting kit-8 was used according to the manufacturer’s protocol. Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays.
  4. Cell viability was evaluated by the absorbance of formazan from each well, and 100% cell viability was calculated from the wells without peptides.
  5. The results are presented as the mean and standard deviation obtained from 5 samples.