Cell penetrating peptides and methods including the same

Abstract

The present disclosure provides chimeric cell-penetrating polypeptides, barcodes, conjugates comprising a cargo moiety and a chimeric cell-penetrating polypeptide described herein, alone or in combination with a barcode of the present technology, and methods of using the conjugates to monitor delivery of the cargo moiety into target tissues or cells.

Description

Atty. Dkt. No.: 115872-3199 CELL PENETRATING PEPTIDES AND METHODS INCLUDING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 559,731, filed February 29, 2024, the contents of which are incorporated herein by reference in their entireties. TECHNICAL FIELD

[0002] The present disclosure relates to chimeric cell-penetrating polypeptides, barcodes, conjugates comprising a cargo moiety and a chimeric cell-penetrating polypeptide described herein, alone or in combination with a barcode of the present technology, and methods of using the conjugates to monitor delivery of the cargo moiety into target tissues or cells. BACKGROUND

[0003] Macromolecular drugs have emerged as promising treatments as a complement or alternative to small molecules for severe diseases including neurodegenerative disorders and cancers. Macromolecules in general and polypeptides specifically offer many advantages to other therapeutics, possessing biochemical features with greater selectivity and specificity, and thus able to target otherwise undruggable factors such as protein-protein interactions.1-5

[0004] However, the relatively large size and polar features of many biological macromolecules makes them impermeable across cellular lipid membranes. The recognition of naturally occurring moieties that confer membrane penetration has led to the development of various cell penetrating peptides (CPPs) and protein transduction domains (PTDs), which are thought to work by membrane pore formation and other interfacial processes, endocytosis with or without involvement of transmembrane proteins, and translocation via inverted micelles followed by their intracellular dissolution.6-10However, the diversity and complexity of these mechanisms have thus far prevented the development of explicit structure-activity relationships, which are necessary for the ultimate design and development of efficient, selective and safe CPPs and PTDs for macromolecular drug delivery.11, 121 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0005] Accordingly, there is an urgent need for new CPPs with improved nuclear and cytoplasmic delivery exceeding hundreds of millions of molecules per human cell, while maintaining minimal membrane disruption and negligible toxicity in vitro. SUMMARY OF THE PRESENT TECHNOLOGY

[0006] In one aspect, the present disclosure provides a peptide barcode comprising an amino acid sequence selected from any one of SEQ ID NOs: 3-98. The peptide barcode may be configured to be detectable by high-resolution mass spectrometry.

[0007] In another aspect, the present disclosure provides a chimeric cell-penetrating polypeptide (CPP) comprising an amino acid sequence selected from the group consisting of GAAIGLAWIPYFGPAAYPRKKRRQRRR (SEQ ID NO: 101), IYNGWYAYGRKKRRQRRR (SEQ ID NO: 102), KLALKLALKALKAALKLAGCYGRKKRRQRRR (SEQ ID NO: 103), and RLALRLALRALRAALRLAGCYGRKKRRQRRR (SEQ ID NO: 104). The CPP may be linked to a peptide barcode. In certain embodiments of the CPP of the present technology, the peptide barcode comprises an amino acid sequence selected from any one of SEQ ID NOs: 3-98. Additionally or alternatively, in some embodiments, the peptide barcode is linked to the N- terminus of the CPP, the C-terminus of the CPP, or a side chain of an amino acid residue of the CPP.

[0008] In yet another aspect, the present disclosure provides a conjugate comprising any and all embodiments of the CPP described herein and a cargo moiety. The cargo moiety may be coupled to the N-terminus of the CPP, the C-terminus of the CPP, or a side chain of an amino acid residue of the CPP. In some embodiments, the CPP is located between the peptide barcode and the cargo moiety. In other embodiments, the peptide barcode is located between the CPP and the cargo moiety. In alternate embodiments, the cargo moiety is located between the peptide barcode and the CPP. Additionally or alternatively, in some embodiments of the conjugates disclosed herein, the cargo moiety comprise a detectable moiety, a therapeutic moiety and / or a targeting moiety. The therapeutic moiety may comprise a small molecule drug, a nucleic acid, a peptidomimetic, an enzyme, an antibody, a therapeutic protein, a receptor agonist, a receptor antagonist, an anticancer agent, an antiviral agent, an antimicrobial agent, an anti-inflammatory 2 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 agent, a protein inhibitor, an immunosuppressive agent, an anesthetic, or any combination thereof.

[0009] In one aspect, the present disclosure provides a kit comprising one or more peptide barcodes comprising an amino acid sequence selected from any one of SEQ ID NOs: 3-98, and instructions for using the same to quantify macromolecules in a biological sample via high- resolution mass spectrometry. The kit may further comprise one or more cell-penetrating peptides and instructions for linking the one or more cell-penetrating peptides to the one or more peptide barcodes. In some embodiments, the biological sample comprises blood, plasma, saliva, urine, serum, CSF, or biopsied tissue.

[0010] In another aspect, the present disclosure provides a kit comprising one or more chimeric cell-penetrating polypeptides (CPPs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 101-104, and instructions for linking the same to a cargo molecule that is configured to be delivered to target tissues or target cells. The kit may further comprise one or more peptide barcodes and instructions for linking the one or more peptide barcodes to the one or more CPPs.

[0011] In yet another aspect, the present disclosure provides a kit comprising at least one CPP of the present technology, at least one peptide barcode of the present technology, and instructions for using the same to monitor delivery of macromolecules in a biological sample or a subject. In some embodiments, wherein the macromolecules comprise one or more of drugs, small molecules, nucleic acids, peptidomimetics, peptides or polypeptides. The biological sample may comprises blood, plasma, saliva, urine, serum, CSF, or biopsied tissue.

[0012] In one aspect, the present disclosure provides a method for delivering a cargo to target tissues or target cells in a subject comprising administering to the subject an effective amount of any and all embodiments of the conjugates described herein. The target tissues or target cells may comprise hematopoietic cells, mesenchymal cells, endothelial cells or epithelial cells. In some embodiments, the target tissues or target cells comprises tissues or cells from bone marrow, colon, rectum, eye, gall bladder, kidney, mouth, larynx, esophagus, stomach, 3 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 intestine, lung, testis, ovary, cervix, brain, liver, skin, spinal cord, thyroid, vagina, vulva, uterus, or muscle.

[0013] In one aspect, the present disclosure provides a method for delivering a cargo to target tissues or target cells in a subject comprising administering to the subject an effective amount of any and all embodiments of the conjugates described herein. The target tissues or target cells may comprise hematopoietic cells, mesenchymal cells, endothelial cells or epithelial cells. In some embodiments, the target tissues or target cells comprises tissues or cells from bone marrow, colon, rectum, eye, gall bladder, kidney, mouth, larynx, esophagus, stomach, intestine, lung, testis, ovary, cervix, brain, liver, skin, spinal cord, thyroid, vagina, vulva, uterus, or muscle.

[0014] In one aspect, the present disclosure provides a method for quantifying macromolecules in a biological sample via high-resolution mass spectrometry comprising (a) coupling macromolecules to a barcoded cell-penetrating peptide, wherein the barcoded cell- penetrating peptide comprises at least one peptide barcode of the present technology to generate a plurality of barcoded macromolecules; (b) incubating a biological sample with the plurality of barcoded macromolecules for a specified period of time; (c) performing cell fractionation on the incubated biological sample to obtain a plurality of fractions including a nuclear fraction and a cytoplasmic fraction; (d) performing high-resolution mass spectrometry on the plurality of fractions; and (e) quantifying absolute abundance of the barcoded macromolecules in the plurality of fractions based on calibration curves generated from the barcoded cell-penetrating peptide in a reference sample. The biological sample and the reference sample may be the same sample type. In some embodiments, the reference sample comprises cell lysate from target cells or target tissues. Additionally or alternatively, in some embodiments, the target tissues or target cells comprise hematopoietic cells, mesenchymal cells, endothelial cells or epithelial cells. Examples of target tissues or target cells include tissues or cells derived from bone marrow, colon, rectum, eye, gall bladder, kidney, mouth, larynx, esophagus, stomach, intestine, lung, testis, ovary, cervix, brain, liver, skin, spinal cord, thyroid, vagina, vulva, uterus, or muscle. 4 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0015] In another aspect, the present disclosure provides a method for selecting a cell- penetrating peptide for quantifying macromolecules in a biological sample via high-resolution mass spectrometry comprising (a) coupling a plurality of cell-penetrating peptides to at least one peptide barcode of the present technology to generate a plurality of barcoded cell-penetrating peptides; (b) incubating each barcoded cell-penetrating peptide with a biological sample for a specified period of time; (c) performing cell fractionation on the incubated biological sample of step (b) to obtain a plurality of fractions including a nuclear fraction and a cytoplasmic fraction; (d) performing high-resolution mass spectrometry on the plurality of fractions of step (c); (e) quantifying mass spectrometry signals detected in step (d) for each barcoded cell-penetrating peptide; and (f) selecting a cell-penetrating peptide based on its mass spectrometry signals in the nuclear fraction and the cytoplasmic fraction.

[0016] In some embodiments, the specified period of time ranges from 10 minutes to 72 hours. Additionally or alternatively, in some embodiments, the specified period of time ranges from 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 12.5 hours, 13 hours, 13.5 hours, 14 hours, 14.5 hours, 15 hours, 15.5 hours, 16 hours, 16.5 hours, 17 hours, 17.5 hours, 18 hours, 18.5 hours, 19 hours, 19.5 hours, 20 hours, 20.5 hours, 21 hours, 21.5 hours, 22 hours, 22.5 hours, 23 hours, 23.5 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours.

[0017] In any of the preceding embodiments of the methods disclosed herein, the macromolecules comprise one or more of drugs, small molecules, nucleic acids, peptidomimetics, peptides or polypeptides. Additionally or alternatively, in some embodiments, the biological sample is obtained from a subject. The subject may be healthy or is diagnosed with or at risk for a disease or condition.

[0018] In yet another aspect, the present disclosure provides a method for tracking delivery of cargo molecules in a subject in real-time comprising (a) coupling cargo molecules to a barcoded cell-penetrating peptide, wherein the barcoded cell-penetrating peptide comprises at least one peptide barcode of the present technology to generate a plurality of barcoded cargo 5 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 molecules; (b) administering the plurality of barcoded cargo molecules to the subject; (c) obtaining a biological sample from the subject at one or more time periods post administration of the barcoded cargo molecules, wherein the biological sample comprises target cells or tissues; (d) performing cell fractionation on the biological sample from the one or more time periods to obtain a plurality of fractions including nuclear fractions and cytoplasmic fractions; (e) performing high-resolution mass spectrometry on the plurality of fractions; and (f) determining real-time delivery of cargo molecules in the subject by identifying / quantifying the mass spectrometry signals of the plurality of barcoded cargo molecules in the nuclear fractions and the cytoplasmic fractions. The subject may be healthy or is diagnosed with or at risk for a disease or condition.

[0019] In some embodiments, the one or more time periods range from at least 1 minute to at least 72 hours post administration of the barcoded cargo molecules. Additionally or alternatively, in some embodiments, the one or more time periods range from at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, at least 10.5 hours, at least 11 hours, at least 12.5 hours, at least 13 hours, at least 13.5 hours, at least 14 hours, at least 14.5 hours, at least 15 hours, at least 15.5 hours, at least 16 hours, at least 16.5 hours, at least 17 hours, at least 17.5 hours, at least 18 hours, at least 18.5 hours, at least 19 hours, at least 19.5 hours, at least 20 hours, at least 20.5 hours, at least 21 hours, at least 21.5 hours, at least 22 hours, at least 22.5 hours, at least 23 hours, at least 23.5 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours.

[0020] Additionally or alternatively, in certain embodiments, the cargo molecules comprise one or more of drugs, small molecules, nucleic acids, peptidomimetics, peptides, or polypeptides.

[0021] In any and all embodiments of the methods disclosed herein, the cell fractionation is achieved using digitonin and sucrose density sedimentation. In any and all embodiments of the 6 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 methods disclosed herein, the biological sample comprises blood, plasma, saliva, urine, serum, CSF, or biopsied tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGs.1A-1E show how the BarcodeBabel algorithm generates unique peptide barcodes that are readily detectable by high-resolution mass spectrometry. FIG.1A: Schematic of BarcodeBabel to generate barcode libraries. FIGs.1B-1D: Signal-response functions for the library of 96 designed barcodes relating the measured mass spectrometric ion intensities to the physical abundance of peptides (See FIG.10) using SIM (FIG.1B), DDA (FIG.1C), and PRM (FIG.1D). MS1 XIC (extracted ion chromatogram) used for SIM and DDA, and MS2 XIC used for PRM. FIG.1E: Slopes (MS signal intensity / amol of barcodes) of the signal-response functions of each barcode obtained from SIM, DDA, and PRM.

[0023] FIG.2 shows the schematic of the PeptideBabel algorithm, which generates bioactive peptide sequences using Markov chain Monte Carlo sampling with permutation of seed sequences (sequence 0) mapped to a density function. PeptideBabel implements a random-walk Metropolis-Hastings algorithm, accepting steps moving up and rejecting steps moving down the density function using user-defined probability functions. The model parameters (θ) and proposed parameters (z) are based on k-mer space that indicates sequence complexity or the physiochemical space (e.g., sequence length, isoelectric point, secondary structure propensity, and hydrophobicity). The figure is based on Jin et al.36

[0024] FIGs.3A-3F show membrane destabilization and cytotoxicity measurements of CPPs. FIG.3A: LDH release of Huh-7 cells after 3-hour incubation of barcoded CPPs. FIG. 3B: Cell viability of Huh-7 cells after 3-hour incubation of barcoded CPPs. FIG.3C: EC50values as a function of AUC of the LDH release measurements. FIG.3D: EC50 values as a function of AUC of the cell viability. FIG.3E: EC50values of LDH release as a function of EC50values of cell viability. FIG.3F: AUC of LDH release as a function of the AUC of cell viability. Symbols and whiskers represent mean and standard deviation values of three biologic replicates, respectively. 7 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0025] FIGs.4A-4B show efficient cell fractionation achieved using digitonin and sucrose density sedimentation. FIG.4A: Representative Western blot with cytoplasmic (Cyt) marker GAPDH, and nuclear (Nu) markers Lamin B1 and Histone H3, as a function of various numbers of freeze-thaw cycles (left), time of treatment with 0.1% digitonin (DGT), and time of treatment with 0.03% DGT, as indicated. FIG.4B: Fluorescence densitometry quantitation of 0.1% DGT- treated cells of Histone 3 abundance (black) and GAPDH abundance (red) in nuclear (Nuc) and cytoplasmic (Cyto) fractions. Bars and whiskers represent mean and standard deviation values of three biologic replicates, respectively.

[0026] FIGs.5A-5C show barcoding and screening strategy results for absolute quantitation of CPP penetration. FIG.5A: Schematic of the cell treatment, fractionation, and LC-MS analysis. FIG.5B: Signal-response function of representative barcode 91, showing MS2 ion current intensity as a function of peptide abundance. Solid line indicates linear fit; dashed line indicates baseline noise value from untreated cell lysate (See FIG.20 for calibration curves for peptide barcodes). FIG.5C: Schematic for data analysis to calculate extracted ion chromatograms, normalized for variation in ionization efficiency using PRTC reference standards and background noise from untreated control samples.

[0027] FIGs.6A-6C show barcoded TAT-P-Ebola peptides exhibit efficient cellular penetration without apparent membrane disruption and cytotoxicity in vitro. FIG.6A: LDH release measurements of Kasumi-1 cells upon 3-hour incubation with barcoded TAT-P-Ebola peptides with various barcodes, as indicated. FIG.6B: Cell viability of Kasumi-1 cells upon 3- hour incubation with barcoded TAT-P-Ebola peptides with various barcodes, as indicated. FIG. 6C: Abundance of barcoded TAT-P-Ebola CPPs in nuclear and cytoplasmic fractions upon 1 mM treatment for 3 hours. Symbols and whiskers indicate mean and standard deviation values of three biologic replicates, respectively.

[0028] FIGs.7A-7D show chimeric CPPs exhibit improved cell type-specific subcellular penetration. FIG.7A: Abundance of barcoded CPPs in nuclear and cytoplasmic fractions of Huh-7 cells upon 3-hour treatment. TAT (black) serves as conventional cationic CPP. badTAT (pink) serves as non-penetrating negative control. FIG.7B: Abundance of barcoded CPPs in 8 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 nuclear and cytoplasmic fractions of Huh-7 cells upon 24-hour treatment. FIGs.7C-7D: Abundance of barcoded KLAL-TAT CPP (FIG.7C) and TAT-P-Ebola CPP (FIG.7D) in nuclear and cytoplasmic fractions of HUVEC, Kasumi-1, Huh-7, and HEK293T cells, as indicated, upon 3-hour treatment. Symbols and whiskers represent mean and standard deviation values for three biologic replicates, respectively. There were no confounder effects for barcodes across all tested cell lines.

[0029] FIG.8 shows a schematic for peptide barcoding and screening for profiling of bioactive macromolecules using BarcodeBabel and PeptideBabel. The subcellular delivery of specific CPPs is deconvoluted using quantitative mass spectrometry of peptide barcodes.

[0030] FIGs.9A-9D show the design strategy and design parameters of BarcodeBabel. A list of 175,579 tryptic peptides of length 5 to 15 residues was queried from the proteometools.org database. Peptide properties were calculated using Biopython tools. FIG.9A: Determining hydrophobicity scores. Calculated Kyte-Doolittle hydrophobicity scores of maximally hydrophobic 5-residue window in tryptic peptides, versus reported proportional retention time from proteometools.org on a linear gradient of 4-35% acetonitrile with 5% DMSO 0.1% formic acid in water.0 = start of gradient, 1 = end of gradient.97% of peptides fall in hydrophobicity range -1.5 to 3.5 and retention range 0.2 to 1.0. FIG.9B: Retention time ranges of 175,579 tryptic peptides of length 5 to 15. Majority of peptides in retention time 0.2 to 0.8. FIG.9C: Setting optimal hydrophobicity score parameters. In retention range 0.2 to 0.8, hydrophobicity range is -0.5 to 2.5 to stay within 1 standard deviation at each hydrophobicity score. FIG.9D: Large numbers of unique BarcodeBabel peptides could be generated in a range of m / z bins.

[0031] FIG.10 shows the pilot 96 barcode sequences designed by BarcodeBabel (B49- B144).

[0032] FIGs.11A-11C show the workflow to identify candidate CPPs by cataloguing viral peptide sequences. FIG.11A: Summary of search. 129 human viral pathogens from ViralZone (https: / / viralzone.expasy.org / ) were searched for presence of known penetration domains per UniProt annotations and literature search. Both full domains and peptide sequences were catalogued. FIGs.11B-11C: Peptide properties. Isoelectric point (Biopython), hydrophobicity 9 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 (Kite-Doolittle scale, Biopython, median of 5-mer windows), and length are shown here for viral peptides as well as previously catalogued peptides from the CPPsite2.0 (http: / / crdd.osdd.net / raghava / cppsite / ) compendium of cell penetration domains.

[0033] FIG.12 shows the CPP candidates chosen for chemical synthesis and cellular delivery assay.

[0034] FIGs.13A-13D show the physiochemical properties of the 96 barcode library for m / z(+2) (FIG.13A) net charge (FIG.13B) isoelectric point (FIG.13C) and hydropathicity (FIG.13D). The barcodes selected for chemical synthesis and cellular delivery assay are highlighted in orange. GRAVY: Grand Average of Hydropathy.

[0035] FIG.14 shows representative LC chromatogram and MS spectrum of the synthetic barcoded CPPs. The LC chromatogram and MS spectrum shown confirmed the successful synthesis of TAT-P-Ebola barcoded with barcode 121. The MW of barcode(121)-TAT-P-Ebola is 4351 Da. Adduct between the target and TFA (trifluoroacetic acid) is present since TFA is used in the LC mobile phases.

[0036] FIG.15 shows LDH release and cell viability profiles of the barcoded CPPs using four different cell lines after incubation for 3 hours. The barcoded-CPPs demonstrated distinct cytotoxicities within the same cell line and across different cell lines, as revealed by LDH release assay and CellTiter-Glo luminescent assay. From top to bottom are LDH release (left panel) and cell viability (right panel) data shown for Kasumi-1, HEK293T, Huh-7, and HUVEC cells, respectively, after incubation for 3 hours at varying concentrations.

[0037] FIG.16 shows LDH release and cell viability profiles of the barcoded CPPs using four different cell lines after incubation for 24 hours. The barcoded-CPPs demonstrated distinct cytotoxicities within the same cell line and across different cell lines, as revealed by LDH release assay and CellTiter-Glo luminescent assay. From top to bottom are LDH release (left panel) and Cell viability (right panel) data shown for Kasumi-1, HEK293T, Huh-7, and HUVEC cells, respectively, after incubation for 24 hours at varying concentrations.

[0038] FIG.17 shows the physiochemical methods tested for cell fractionation efficiency. 10 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0039] FIGs.18A-18C show evaluations of the cell fractionation efficiency of various physical and chemical methods. FIG.18A: Phase contrast micrographs at 4x magnification. FIG.18B: Phase contrast micrographs images at 20x magnification. FIG.18C: Western blot with cytoplasmic marker GAPDH and nuclear markers Lamin B1 and Histone H3. The control (no lysis) sample contains ~0.8 M Kasumi-1 cells in 300 µL hypotonic buffer, while all other samples contain ~1.8 M Kasumi-1 cells in 300 µL hypotonic buffer.

[0040] FIG.19 shows experimental conditions tested for freeze-thaw and digitonin-based cell fractionation methods.

[0041] FIG.20 shows calibration curves of all the barcodes used in the cellular delivery assays. Dashed lines represent linear fits. The MS2 ion current signals of the baseline control samples (untreated cell lysate) are shown as solid lines. The CPPs corresponding to each barcode is as follows: badTAT for barcode 55, TAT for barcode 64, KLAL-TAT for barcode 82, P14 for barcode 91, TAT-G-EBV for barcode 107, RLAL-TAT for barcode 108, TAT-P-Ebola for barcode 121, and TAT-P-Ebola for barcode 81.

[0042] FIGs.21A-21F show cytotoxicity profiles and cellular delivery of barcoded TAT-P- Ebola peptides. FIGs.21A-21D: Cytotoxicity of barcoded TAT-P-Ebola peptides on Kasumi-1 cells. FIGs.21A-21B show LDH release assays after incubation for 3 and 24 hours. FIGs.21C- 21D show CellTiter-Glo luminescent assay after incubation for 3 and 24 hours. FIGs.21E-21F: Relative and absolute cellular delivery of the barcoded TAT-P-Ebola. FIG.21E: The extracted MS2 ion current signal for each barcode. FIG.21F: Quantitative cellular delivery of the barcoded TAT-P-Ebola in Kasumi-1 cells.

[0043] FIGs.22A-22G show extracted MS2 ion current signals for each barcode in each cell fraction across four different cell lines as indicated in each panel. The MS2 ion current signals of the baseline control sample (untreated cell lysate) with a 10% signal deviation were shown in black and red horizontal lines. The reference line with extra 10% of the baseline readings accounts for the slight batch-to-batch signal fluctuations in MS spectra. 11 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0044] FIGs.23A-23D show quantitative cellular delivery of the barcoded CPPs in four different cell lines (FIG.23A Kasumi-1; FIG.23B HEK293T cells; FIG.23C Huh-7 cells; and FIG.23D HUVEC cells). DETAILED DESCRIPTION

[0045] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0046] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well- known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)). 12 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0047] Disclosed herein are peptide barcodes and chimeric CPPs optimized for screening and analysis by quantitative mass spectrometry. The chimeric CPPs of the present technology are composed of unrelated CPP sequences that do not exist in nature and exhibit distinct physicochemical properties as estimated by their m / z, net charge, and hydrophobicity values. The chimeric CPPs described herein exhibit improved nuclear and cytoplasmic delivery exceeding hundreds of millions of molecules per human cell, while maintaining minimal membrane disruption and negligible toxicity in vitro.

[0048] Without wishing to be bound by theory, the peptide barcodes of the present technology generate specific signature ions, which facilitate efficient monitoring / tracking via high-resolution mass spectrometry. Further, the Examples described herein demonstrate that the peptide barcodes of the present technology modulated the apparent CPP activity, suggesting that peptide barcodes may themselves be incorporated into the design of bioactive molecules. This also indicates that high-throughput screens should utilize multiple independent barcodes to discern specific biological activities, as is practiced with nucleic acid barcoding and other high- throughput technologies. Definitions

[0049] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0050] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). 13 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0051] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally, or topically. Administration includes self-administration and the administration by another.

[0052] As used herein, the term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0053] As used herein, “cell fractionation” refers to the process of separating subcellular components and organelles into distinct fractions, while preserving individual functions of each component. The process involves homogenizing tissue in a buffered isotonic solution to obtain a cell homogenate, filtering the cell homogenate to remove debris, and purifying the cell homogenate using differential centrifugation to obtain a plurality of cell fractions. Mechanisms for homogenization include grinding, mincing, chopping, pressure changes, osmotic shock, freeze-thawing, and ultrasound.

[0054] As used herein, the term “conjugated” refers to the association of two molecules by any method known to those in the art. Suitable types of associations include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for instance, hydrogen bonds, dipolar interactions, van der Waal forces, electrostatic interactions, hydrophobic interactions and aromatic stacking.

[0055] By “binding affinity” is meant the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y can generally be 14 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 represented by the dissociation constant (KD). Affinity can be measured by standard methods known in the art, including those described herein.

[0056] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease or condition, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0057] As used herein, the terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit” means a decrease by a statistically significant amount. For avoidance of doubt, “decrease”, “reduced”, “reduction”, “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0058] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a “therapeutically effective amount” of a composition refers to 15 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0059] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if required for proper expression and function.

[0060] As used herein, “expression control sequence” or “regulatory region” of a nucleic acid molecule means a cis- acting nucleotide sequence that influences expression, positively or negatively, of an operatively linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.

[0061] Particular examples of gene regulatory regions are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more. 16 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0062] Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.

[0063] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0064] As used herein, the terms “increased”, “increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount. For the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10- fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0065] As used herein, the term “heterologous nucleic acid molecule or polypeptide” refers to a nucleic acid molecule (e.g ., a cDNA, DNA or RNA molecule) or polypeptide that is either not normally expressed or is expressed at an aberrant level in a cell or sample obtained from a cell. This nucleic acid can be from another organism, or it can be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

[0066] As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human. 17 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0067] As used herein, "operably linked" with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, nucleic acid encoding a leader peptide can be operably linked to nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide effects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.

[0068] As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20thedition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0069] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double- stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA 18 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0070] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.

[0071] As used herein, “prevention”, “prevent”, or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample. As used herein, preventing a disease or condition, includes preventing or delaying the initiation of symptoms of the disease or condition or preventing a recurrence of one or more signs or symptoms of the disease or condition.

[0072] As used herein, the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0073] As used herein, the term “sample” refers to clinical samples obtained from a subject. Biological samples may include tissues, cells, protein or membrane extracts of cells, mucus, sputum, bone marrow, bronchial alveolar lavage (BAL), bronchial wash (BW), and biological 19 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids (blood, plasma, saliva, urine, serum etc.) present within a subject.

[0074] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0075] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0076] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0077] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0078] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0079] It is also to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean “substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically 20 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0080] As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins or RNAs can be expressed when the vector is introduced into an appropriate host cell. The vector is used to introduce the nucleic acid encoding the polypeptide or RNA into the host cell for amplification of the nucleic acid or for expression / display of the polypeptide or RNA encoded by the nucleic acid. As used herein, a vector also includes "virus vectors" or "viral vectors." Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells. Peptide Barcodes and CPPs

[0081] In one aspect, the present disclosure provides a peptide barcode comprising an amino acid sequence selected from any one of SEQ ID NOs: 3-98. The peptide barcode may be configured to be detectable by high-resolution mass spectrometry. Without wishing to be bound by theory, the peptide barcodes of the present technology generate specific signature ions, which facilitate efficient monitoring / tracking via high-resolution mass spectrometry.

[0082] Also disclosed herein are chimeric cell penetrating peptides and conjugates comprising a cell penetrating peptide moiety and a cargo moiety. The cargo moiety can comprise one or more detectable moieties, one or more therapeutic moieties, one or more targeting moieties, or any combination thereof. The cargo moiety can be attached to the CPP moiety at its amino terminus, its carboxy terminus, or at a side chain of any of the amino acids of the CPP moiety.

[0083] The CPPs may comprise an amino acid sequence selected from among GAAIGLAWIPYFGPAAYPRKKRRQRRR (SEQ ID NO: 101), IYNGWYAYGRKKRRQRRR (SEQ ID NO: 102), KLALKLALKALKAALKLAGCYGRKKRRQRRR (SEQ ID NO: 103), and RLALRLALRALRAALRLAGCYGRKKRRQRRR (SEQ ID NO: 104).

[0084] As used herein “cellular uptake efficiency” refers to the ability of a CPP sequence to traverse a cell membrane. In some embodiments, cellular uptake of the CPP may or may not be 21 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 dependent on a receptor or a cell type. In some embodiments, uptake efficiency can be improved by at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 4.5 fold, at least about 5 fold, at least about 5.5 fold, at least about 6 fold, at least about 6.5 fold, at least about 7 fold, at least about 7.5 fold, at least about 8 fold, at least about 8.5 fold, at least about 9 fold, at 9.5 fold, or at least about 10 fold. In other embodiments, the uptake efficiency can be improved within the range of from about 1.5 fold to about 10 fold, or about 2 fold to about 10 fold, or about 2 fold to about 9.5 fold, or about 2 fold to about 9 fold, or about 2 fold to about 8.5 fold, or about 2 fold to about 8 fold, or about 2 fold to about 7.5 fold, or about 2 fold to about 7 fold, or about 2 fold to about 6.5 fold, or about 2 fold to about 6 fold, or about 2.5 fold to about 7 fold, or about 3 fold to about 7 fold, or about 3.5 fold to about 7 fold, or about 4 to about 7, or about 4.5 fold to about 7 fold, or about 5 fold to about 7 fold, or about 5.5 fold to about 7 fold, or about 6 fold to about 7 fold.

[0085] Additionally or alternatively, in some embodiments, the CPPs disclosed herein may comprise an N-terminal acetyl group and / or a C-terminal amide group.

[0086] Also disclosed herein are pharmaceutically-acceptable salts of the CPPs disclosed herein. Pharmaceutically-acceptable salts include salts of the disclosed compounds that are prepared with acids or bases, depending on the particular substituents found on the compounds. Under conditions where the compounds disclosed herein are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts can be appropriate. Examples of pharmaceutically-acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salt. Examples of physiologically-acceptable acid addition salts include hydrochloric, hydrobromic, nitric, phosphoric, carbonic, sulfuric, and organic acids like acetic, propionic, benzoic, succinic, fumaric, mandelic, oxalic, citric, tartaric, malonic, ascorbic, alpha-ketoglutaric, alpha-glycophosphoric, maleic, tosyl acid, methanesulfonic, and the like. Thus, disclosed herein are the hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate, acetate, propionate, benzoate, succinate, fumarate, mandelate, oxalate, citrate, tartarate, malonate, ascorbate, alpha-ketoglutarate, alpha-glycophosphate, maleate, tosylate, and mesylate salts. Pharmaceutically acceptable salts of a compound can be obtained using standard 22 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

[0087] Additionally or alternatively, in some embodiments, the CPPs of the present technology may be operably linked to one or more peptide barcodes. In some embodiments, the CPP is located between the peptide barcode and the cargo moiety. In other embodiments, the peptide barcode is located between the CPP and the cargo moiety. In alternate embodiments, the cargo moiety is located between the peptide barcode and the CPP.

[0088] Additionally or alternatively, in some embodiments, the peptide barcodes are configured to be detectable by high-resolution mass spectrometry. In certain embodiments, the one or more peptide barcodes lack internal amino acids that may interfere with correct enzymatic cleavage and consistent mass spectrometric detection or fragmentation, such as lysine, arginine, histidine, cysteine, methionine, proline, and isoleucine. Additionally or alternatively, in some embodiments, the one or more peptide barcodes comprise an amino acid sequence selected from any one of SEQ ID NOs: 3-98. In some embodiments, the peptide barcode can be linked to the CPP moiety at its amino terminus, its carboxy terminus, or at a side chain of any of the amino acids of the CPP moiety. Cargo Moiety

[0089] The cargo moiety can comprise any cargo of interest, for example a linker moiety, a detectable moiety, a therapeutic moiety, a targeting moiety, and the like, or any combination thereof. In some examples, the cargo moiety can comprise one or more additional amino acids (e.g., K, UK, TRV); a linker (e.g., bifunctional linker LC-SMCC); coenzyme A; phosphocoumaryl amino propionic acid (pCAP); 8-amino-3,6-dioxaoctanoic acid (miniPEG); L- 2,3-diaminopropionic acid (Dap or J); L-β-naphthylalanine; L-pipecolic acid (Pip); sarcosine; trimesic acid; 7-amino-4-methylcourmarin (Amc); fluorescein isothiocyanate (FITC); L-2- naphthylalanine; norleucine; 2-aminobutyric acid; Rhodamine B (Rho); Dexamethasone (DEX); or combinations thereof. 23 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0090] In some examples the cargo moiety can comprise any of those listed in Table A, or derivatives or combinations thereof. Table A: Exemplary cargo moieties RRRRR (SEQ ID NO: 106) AAAAA (SEQ ID NO: 107) FFFF (SEQ ID NO: 108) DE(pCAP)LI (SEQ ID NO: 109) AAAAAAA (SEQ ID NO: 110) RARAR (SEQ ID NO: 111) DADAD (SEQ ID NO: 112) DΩUD (SEQ ID NO: 113) UTRV (SEQ ID NO: 114) *pCAP, phosphocoumaryl amino propionic acid; Ω, norleucine; U, 2-aminobutyric acid. Detectable Moiety

[0091] The detectable moiety can comprise any detectable label. Examples of suitable detectable labels include, but are not limited to, a UV-Vis label, a near-infrared label, a luminescent group, a phosphorescent group, a magnetic spin resonance label, a photosensitizer, a photocleavable moiety, a chelating center, a heavy atom, a radioactive isotope, an isotope detectable spin resonance label, a paramagnetic moiety, a chromophore, or any combination thereof. In some embodiments, the label is detectable without the addition of further reagents.

[0092] In some embodiments, the detectable moiety is a biocompatible detectable moiety, such that the CPPs can be suitable for use in a variety of biological applications. “Biocompatible” and “biologically compatible”, as used herein, generally refer to compounds that are, along with any metabolites or degradation products thereof, generally non-toxic to cells 24 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 and tissues, and which do not cause any significant adverse effects to cells and tissues when cells and tissues are incubated (e.g., cultured) in their presence.

[0093] The detectable moiety can contain a luminophore such as a fluorescent label or near- infrared label. Examples of suitable luminophores include, but are not limited to, metal porphyrins; benzoporphyrins; azabenzoporphyrine; napthoporphyrin; phthalocyanine; polycyclic aromatic hydrocarbons such as perylene, perylene diimine, pyrenes; azo dyes; xanthene dyes; boron dipyoromethene, aza-boron dipyoromethene, cyanine dyes, metal-ligand complex such as bipyridine, bipyridyls, phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium; acridine, oxazine derivatives such as benzophenoxazine; aza-annulene, squaraine; 8- hydroxyquinoline, polymethines, luminescent producing nanoparticle, such as quantum dots, nanocrystals; carbostyril; terbium complex; inorganic phosphor; ionophore such as crown ethers affiliated or derivatized dyes; or combinations thereof. Specific examples of suitable luminophores include, but are not limited to, Pd (II) octaethylporphyrin; Pt (II)- octaethylporphyrin; Pd (II) tetraphenylporphyrin; Pt (II) tetraphenylporphyrin; Pd (II) meso- tetraphenylporphyrin tetrabenzoporphine; Pt (II) meso-tetrapheny metrylbenzoporphyrin; Pd (II) octaethylporphyrin ketone; Pt (II) octaethylporphyrin ketone; Pd (II) meso- tetra(pentafluorophenyl)porphyrin; Pt (II) meso-tetra (pentafluorophenyl) porphyrin; Ru (II) tris(4,7-diphenyl-1,10-phenanthroline) (Ru (dpp)3); Ru (II) tris(1,10-phenanthroline) (Ru(phen)3), tris(2,2′-bipyridine)ruthenium (II) chloride hexahydrate (Ru(bpy)3); erythrosine B; fluorescein; fluorescein isothiocyanate (FITC); eosin; iridium (III) ((N-methyl-benzimidazol-2- yl)-7-(diethylamino)-coumarin)); indium (III) ((benzothiazol-2-yl)-7-(diethylamino)-coumarin))- 2-(acetylacetonate); Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow; Texas Red; rhodamine B; rhodamine 6G; sulfur rhodamine; m-cresol; thymol blue; xylenol blue; cresol red; chlorophenol blue; bromocresol green; bromcresol red; bromothymol blue; Cy2; a Cy3; a Cy5; a Cy5.5; Cy7; 4-nitirophenol; alizarin; phenolphthalein; o-cresolphthalein; chlorophenol red; calmagite; bromo-xylenol; phenol red; neutral red; nitrazine; 3,4,5,6- tetrabromphenolphtalein; congo red; fluorescein; eosin; 2′,7′-dichlorofluorescein; 5(6)-carboxy- fluorecsein; carboxynaphthofluorescein; 8-hydroxypyrene-1,3,6-trisulfonic acid; semi- naphthorhodafluor; semi-naphthofluorescein; tris (4,7-diphenyl-1,10-phenanthroline) ruthenium 25 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 (II) dichloride; (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) tetraphenylboron; platinum (II) octaethylporphyin; dialkylcarbocyanine; dioctadecylcycloxacarbocyanine; fluorenylmethyloxycarbonyl chloride; 7-amino-4-methylcourmarin (Amc); green fluorescent protein (GFP); and derivatives or combinations thereof. In some examples, the detectable moiety can comprise Rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4- methylcourmarin (Amc), green fluorescent protein (GFP), or derivatives or combinations thereof.

[0094] The detectable moiety can be attached to the CPP moiety at its amino terminus, its carboxy terminus, or at a side chain of any of the amino acids of the CPP moiety. Therapeutic Moiety

[0095] The CPPs, alone or in combination with the peptide barcodes disclosed herein, can also comprise a therapeutic moiety. In some examples, the cargo moiety comprises a therapeutic moiety. The detectable moiety can be linked to a therapeutic moiety or the detectable moiety can also serve as the therapeutic moiety. Therapeutic moiety refers to a group that when administered to a subject will reduce one or more symptoms of a disease or disorder.

[0096] The therapeutic moiety can comprise a wide variety of drugs, including antagonists, for example enzyme inhibitors, and agonists, for example a transcription factor which results in an increase in the expression of a desirable gene product (although as will be appreciated by those in the art, antagonistic transcription factors can also be used), are all included. In addition, therapeutic moiety includes those agents capable of direct toxicity and / or capable of inducing toxicity towards healthy and / or unhealthy cells in the body. In some embodiments, the therapeutic moiety can be capable of inducing and / or priming the immune system against potential pathogens. The therapeutic moiety can, for example, comprise an anticancer agent, an antibody, antiviral agent, antimicrobial agent, anti-inflammatory agent (e.g., dexamethasone), immunosuppressive agent, anesthetics, therapeutic proteins (e.g., enzymes) or any combination thereof.

[0097] Examples of anticancer agents include, but are not limited to, 13-cis-Retinoic Acid, 2- Amino-6-Mercaptopurine, 2-CdA, 2-Chlorodeoxyadenosine, 5-fluorouracil, 6-Thioguanine, 6- 26 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 Mercaptopurine, Accutane, Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alitretinoin, Alkaban-AQ, Alkeran, All-transretinoic acid, Alpha interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron, Anastrozole, Arabinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, Arsenic trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib, Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar, Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine, Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine, cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine liposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposomal, DaunoXome, Decadron, Delta-Cortef, Deltasone, Denileukin diftitox, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC- Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate, Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim, Floxuridine, Fludara, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron, Lupron Depot, Matulane, Maxidex, Mechlorethamine, - Mechlorethamine Hydrochlorine, Medralone, Medrol, Megace, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate Sodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta, Neumega, Neupogen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-asparaginase, Phenylalanine Mustard, Platinol, Platinol-AQ, Prednisolone, Prednisone, Prelone, Procarbazine, PROCRIT, Proleukin, Prolifeprospan 20 with Carmustine implant, Purinethol, Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alfa-2a), 27 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 Rubex, Rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid, Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon, Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa, Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulating factor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, HMM, Hycamtin, Hydrea, Hydrocort Acetate, Hydrocortisone, Hydrocortisone sodium phosphate, Hydrocortisone sodium succinate, Hydrocortone phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin, Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib mesylate, Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEG conjugate), Interleukin 2, Interleukin-11, Intron A (interferon alfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine, Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, L-PAM, L- Sarcolysin, Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX, Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan, Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR. The therapeutic moiety can also comprise a biopharmaceutical such as, for example, an antibody.

[0098] In some embodiments, the therapeutic moiety can comprise an antiviral agent, such as ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc.

[0099] Examples of antibacterial agents, include but are not limited to, acedapsone; acetosulfone sodium; alamecin; alexidine; amdinocillin; amdinocillin pivoxil; amicycline; amifloxacin; amifloxacin mesylate; amikacin; amikacin sulfate; aminosalicylic acid; aminosalicylate sodium; amoxicillin; amphomycin; ampicillin; ampicillin sodium; apalcillin sodium; apramycin; aspartocin; astromicin sulfate; avilamycin; avoparcin; azithromycin; azlocillin; azlocillin sodium; bacampicillin hydrochloride; bacitracin; bacitracin methylene disalicylate; bacitracin zinc; bambermycins; benzoylpas calcium; berythromycin; betamicin 28 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 sulfate; biapenem; biniramycin; biphenamine hydrochloride; bispyrithione magsulfex; butikacin; butirosin sulfate; capreomycin sulfate; carbadox; carbenicillin disodium; carbenicillin indanyl sodium; carbenicillin phenyl sodium; carbenicillin potassium; carumonam sodium; cefaclor; cefadroxil; cefamandole; cefamandole nafate; cefamandole sodium; cefaparole; cefatrizine; cefazaflur sodium; cefazolin; cefazolin sodium; cefbuperazone; cefdinir; cefepime; cefepime hydrochloride; cefetecol; cefixime; cefmenoxime hydrochloride; cefmetazole; cefmetazole sodium; cefonicid monosodium; cefonicid sodium; cefoperazone sodium; ceforanide; cefotaxime sodium; cefotetan; cefotetan disodium; cefotiam hydrochloride; cefoxitin; cefoxitin sodium; cefpimizole; cefpimizole sodium; cefpiramide; cefpiramide sodium; cefpirome sulfate; cefpodoxime proxetil; cefprozil; cefroxadine; cefsulodin sodium; ceftazidime; ceftibuten; ceftizoxime sodium; ceftriaxone sodium; cefuroxime; cefuroxime axetil; cefuroxime pivoxetil; cefuroxime sodium; cephacetrile sodium; cephalexin; cephalexin hydrochloride; cephaloglycin; cephaloridine; cephalothin sodium; cephapirin sodium; cephradine; cetocycline hydrochloride; cetophenicol; chloramphenicol; chloramphenicol palmitate; chloramphenicol pantothenate complex; chloramphenicol sodium succinate; chlorhexidine phosphanilate; chloroxylenol; chlortetracycline bisulfate; chlortetracycline hydrochloride; cinoxacin; ciprofloxacin; ciprofloxacin hydrochloride; cirolemycin; clarithromycin; clinafloxacin hydrochloride; clindamycin; clindamycin hydrochloride; clindamycin palmitate hydrochloride; clindamycin phosphate; clofazimine; cloxacillin benzathine; cloxacillin sodium; cloxyquin; colistimethate sodium; colistin sulfate; coumermycin; coumermycin sodium; cyclacillin; cycloserine; dalfopristin; dapsone; daptomycin; demeclocycline; demeclocycline hydrochloride; demecycline; denofungin; diaveridine; dicloxacillin; dicloxacillin sodium; dihydrostreptomycin sulfate; dipyrithione; dirithromycin; doxycycline; doxycycline calcium; doxycycline fosfatex; doxycycline hyclate; droxacin sodium; enoxacin; epicillin; epitetracycline hydrochloride; erythromycin; erythromycin acistrate; erythromycin estolate; erythromycin ethylsuccinate; erythromycin gluceptate; erythromycin lactobionate; erythromycin propionate; erythromycin stearate; ethambutol hydrochloride; ethionamide; fleroxacin; floxacillin; fludalanine; flumequine; fosfomycin; fosfomycin tromethamine; fumoxicillin; furazolium chloride; furazolium tartrate; fusidate sodium; fusidic acid; gentamicin sulfate; gloximonam; gramicidin; 29 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 haloprogin; hetacillin; hetacillin potassium; hexedine; ibafloxacin; imipenem; isoconazole; isepamicin; isoniazid; josamycin; kanamycin sulfate; kitasamycin; levofuraltadone; levopropylcillin potassium; lexithromycin; lincomycin; lincomycin hydrochloride; lomefloxacin; Lomefloxacin hydrochloride; lomefloxacin mesylate; loracarbef; mafenide; meclocycline; meclocycline sulfosalicylate; megalomicin potassium phosphate; mequidox; meropenem; methacycline; methacycline hydrochloride; methenamine; methenamine hippurate; methenamine mandelate; methicillin sodium; metioprim; metronidazole hydrochloride; metronidazole phosphate; mezlocillin; mezlocillin sodium; minocycline; minocycline hydrochloride; mirincamycin hydrochloride; monensin; monensin sodiumr; nafcillin sodium; nalidixate sodium; nalidixic acid; natainycin; nebramycin; neomycin palmitate; neomycin sulfate; neomycin undecylenate; netilmicin sulfate; neutramycin; nifuiradene; nifuraldezone; nifuratel; nifuratrone; nifurdazil; nifurimide; nifiupirinol; nifurquinazol; nifurthiazole; nitrocycline; nitrofurantoin; nitromide; norfloxacin; novobiocin sodium; ofloxacin; onnetoprim; oxacillin; oxacillin sodium; oximonam; oximonam sodium; oxolinic acid; oxytetracycline; oxytetracycline calcium; oxytetracycline hydrochloride; paldimycin; parachlorophenol; paulomycin; pefloxacin; pefloxacin mesylate; penamecillin; penicillin G benzathine; penicillin G potassium; penicillin G procaine; penicillin G sodium; penicillin V; penicillin V benzathine; penicillin V hydrabamine; penicillin V potassium; pentizidone sodium; phenyl aminosalicylate; piperacillin sodium; pirbenicillin sodium; piridicillin sodium; pirlimycin hydrochloride; pivampicillin hydrochloride; pivampicillin pamoate; pivampicillin probenate; polymyxin B sulfate; porfiromycin; propikacin; pyrazinamide; pyrithione zinc; quindecamine acetate; quinupristin; racephenicol; ramoplanin; ranimycin; relomycin; repromicin; rifabutin; rifametane; rifamexil; rifamide; rifampin; rifapentine; rifaximin; rolitetracycline; rolitetracycline nitrate; rosaramicin; rosaramicin butyrate; rosaramicin propionate; rosaramicin sodium phosphate; rosaramicin stearate; rosoxacin; roxarsone; roxithromycin; sancycline; sanfetrinem sodium; sarmoxicillin; sarpicillin; scopafungin; sisomicin; sisomicin sulfate; sparfloxacin; spectinomycin hydrochloride; spiramycin; stallimycin hydrochloride; steffimycin; streptomycin sulfate; streptonicozid; sulfabenz; sulfabenzamide; sulfacetamide; sulfacetamide sodium; sulfacytine; sulfadiazine; sulfadiazine sodium; sulfadoxine; sulfalene; sulfamerazine; sulfameter; sulfamethazine; 30 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 sulfamethizole; sulfamethoxazole; sulfamonomethoxine; sulfamoxole; sulfanilate zinc; sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; sulfazamet; sulfisoxazole; sulfisoxazole acetyl; sulfisboxazole diolamine; sulfomyxin; sulopenem; sultamricillin; suncillin sodium; talampicillin hydrochloride; teicoplanin; temafloxacin hydrochloride; temocillin; tetracycline; tetracycline hydrochloride; tetracycline phosphate complex; tetroxoprim; thiamphenicol; thiphencillin potassium; ticarcillin cresyl sodium; ticarcillin disodium; ticarcillin monosodium; ticlatone; tiodonium chloride; tobramycin; tobramycin sulfate; tosufloxacin; trimethoprim; trimethoprim sulfate; trisulfapyrimidines; troleandomycin; trospectomycin sulfate; tyrothricin; vancomycin; vancomycin hydrochloride; virginiamycin; or zorbamycin.

[0100] The therapeutic moiety can be attached to the CPP moiety at its amino terminus, its carboxy terminus, or at a side chain of any of the amino acids of the CPP moiety. In some embodiments, the therapeutic moiety can be attached to the detectable moiety. Targeting Moiety

[0101] In some embodiments, the therapeutic moiety comprises a targeting moiety. The targeting moiety can comprise, for example, a sequence of amino acids that can target one or more enzyme domains. In some examples, the targeting moiety can comprise an inhibitor against an enzyme that can play a role in a disease. In some examples, the targeting moiety may comprise any of those listed in Table B. Table B: Exemplary targeting moieties Abbreviation* Sequence PΘGΛYR Pro-Pip-Gly-F2Pmp-Tyr-Arg (SEQ ID NO: 115) SΘIΛΛR Ser-Pip-Ile-F2Pmp-F2Pmp-Arg (SEQ ID NO: 116) IHIΛIR Ile-His-Ile-F2Pmp-Ile-Arg (SEQ ID NO: 117) AaIΛΘR Ala-(D-Ala)-Ile-F2Pmp-Pip-Arg (SEQ ID NO: 118) ΣSΘΛvR Fpa-Ser-Pip-F2Pmp-(D-Val)-Arg (SEQ ID NO: 119) ΘnPΛAR Pip-(D-Asn)-Pro-F2Pmp-Ala-Arg (SEQ ID NO: 120) TΨPAΛGR Tyr-Phg-Ala-F2Pmp-Gly-Arg (SEQ ID NO: 121) AHIΛaR Ala-His-Ile-F2Pmp-(D-Ala)-Arg (SEQ ID NO: 122) GnGΛpR Gly-(D-Asn)-Gly-F2Pmp-(D-Pro)-Arg (SEQ ID NO: 123) 31 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 fQΘΛIR (D-Phe)-Gln-Pip-F2Pmp-Ile-Arg (SEQ ID NO: 124) SPGΛHR Ser-Pro-Gly-F2Pmp-His-Arg (SEQ ID NO: 125) ΘYIΛHR Pip-Tyr-Ile-F2Pmp-His-Arg (SEQ ID NO: 126) SvPΛHR Ser-(D-Val)-Pro-F2Pmp-His-Arg (SEQ ID NO: 127) AIPΛnR Ala-Ile-Pro-F2Pmp-(D-Asn)-Arg (SEQ ID NO: 128) ΣSIΛQF Fpa-Ser-Ile-F2Pmp-Gln-Phe (SEQ ID NO: 129) AaΨPΛfR Ala-(D-Ala)-Phg-F2Pmp-(D-Phe)-Arg (SEQ ID NO: 130) ntΨΛΨR (D-Asn)-(D-Thr)-Phg-F2Pmp-Phg-Arg (SEQ ID NO: 131) IPΨΛΩR Ile-Pro-Phg-F2Pmp-Nle-Arg (SEQ ID NO: 132) QΘΣΛΘR Gln-Pip-Fpa-F2Pmp-Pip-Arg (SEQ ID NO: 133) nAΣΛGR (D-Asn)-Ala-Fpa-F2Pmp-Gly-Arg (SEQ ID NO: 134) ntYΛAR (D-Asn)-(D-Thr)-Tyr-F2Pmp-Ala-Arg (SEQ ID NO: 135) eAΨΛvR (D-Glu)-Ala-Phg-F2Pmp-(D-Val)-Arg (SEQ ID NO: 136) IvΨΛAR Ile-(D-Val)-Phg-F2Pmp-Ala-Arg (SEQ ID NO: 137) YtΨΛAR Tyr-(D-Thr)-Phg-F2Pmp-Ala-Arg (SEQ ID NO: 138) nΘΨΛIR (D-Asn)-Pip-Phg-F2Pmp-Ile-Arg (SEQ ID NO: 139) ΘnWΛHR Pip-(D-Asn)-Trp-F2Pmp-His-Arg (SEQ ID NO: 140) YΘvΛIR Tyr-Pip-(D-Val)-F2Pmp-Ile-Arg (SEQ ID NO: 141) nSAΛGR (D-Asn)-Ser-(D-Ala)-F2Pmp-Gly-Arg (SEQ ID NO: 142) tnvΛaR (D-Thr)-(D-Asn)-(D-Val)-F2Pmp-(D-Ala)- Arg (SEQ ID NO: 143) ntvΛtR (D-Asn)-(D-Thr)-(D-Val)-F2Pmp-(D-Thr)-Arg (SEQ ID NO: 144) SItΛYR Ser-Ile-(D-Thr)-F2Pmp-Tyr-Arg (SEQ ID NO: 145) nΣnΛlR (D-Asn)-Fpa-(D-Asn)-F2Pmp-(D-Leu)-Arg (SEQ ID NO: 146) YnnΛΩR Tyr-(D-Asn)-(D-Asn)-F2Pmp-Nle-Arg (SEQ ID NO: 147) nYnΛGR (D-Asn)-Tyr-(D-Asn)-F2Pmp-Gly-Arg (SEQ ID NO: 148) AWnΛAR Ala-Trp-(D-Asn)-F2Pmp-Ala-Arg (SEQ ID NO: 149) vtHΛYR (D-Val)-(D-Thr)-His-F2Pmp-Tyr-Arg (SEQ ID NO: 150) PΨHΛΘR Pro-Phg-His-F2Pmp-Pip-Arg (SEQ ID NO: 151) nΨHΛGR (D-Asn)-Phg-His-F2Pmp-Gly-Arg (SEQ ID NO: 152) PAHΛGR Pro-Ala-His-F2Pmp-Gly-Arg (SEQ ID NO: 153) AYHΛIR Ala-Tyr-His-F2Pmp-Ile-Arg (SEQ ID NO: 154) nΘeΛYR (D-Asn)-Pip-(D-Glu)-F2Pmp-Tyr-Arg (SEQ ID NO: 155) vSSΛtR (D-Val)-Ser-Ser-F2Pmp-(D-Thr)-Arg (SEQ ID NO: 156) 32 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 aΞt′ϑΦ′YNK ((D-Ala)-Sar-(D-pThr)-Pp-Nal-Tyr-Gln)-Lys (SEQ ID NO: 157) Tm(aΞt′ϑΦ′RA)Dap Tm((D-Ala)-Sar-(D-pThr)-Pp-Nal-Arg-Ala)-Dap (SEQ ID NO: 158) Tm(aΞt′ϑΦ′RAa)Dap Tm((D-Ala)-Sar-(D-pThr)-Pp-Nal-Arg-Ala-(D-Ala))-Dap (SEQ ID NO: 159) Tm(aΞtϑΦ′RAa)Dap Tm((D-Ala)-Sar-(D-Thr)-Pp-Nal-Arg-Ala-(D- Ala))-Dap (SEQ ID NO: 160) Tm(aΞtaΦ′RAa)Dap Tm((D-Ala)-Sar-(D-Thr)-(D-Ala)-Nal-Arg-Ala-(D-Ala))-Dap (SEQ ID NO: 161) *Fpa, Ξ: L-4-fluorophenylalanine; Pip, Φ: L-homoproline; Nle, Ω: L-norleucine; Phg, Ψ L- phenylglycine; F2Pmp, Λ: L-4-(phosphonodifluoromethyl)phenylalanine; Dap, L-2,3- diaminopropionic acid; Nal, Φ′: L-β-naphthylalanine; Pp, ϑ: L-pipecolic acid; Sar, Ξ: sarcosine; Tm, trimesic acid.

[0102] The targeting moiety and cell penetrating peptide moiety can overlap, that is residues that form the cell penetrating peptide moiety can also be part of the sequence that forms the targeting moiety, and vice a versa. In certain embodiments, the therapeutic moiety can comprise a targeting moiety that can act as an inhibitor against Ras (e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, CAL PDZ, and the like, or combinations thereof. Compositions, Formulations and Modes of Administration

[0103] In vivo application of the disclosed CPPs, alone or in combination with the peptide barcodes disclosed herein, and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed compositions can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Administration of the disclosed CPPs, alone or in combination with the peptide barcodes disclosed herein can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art. 33 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0104] The CPPs and / or the peptide barcodes disclosed herein, and compositions comprising them, can also be administered utilizing liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time. The CPPs and / or the peptide barcodes can also be administered in their salt derivative forms or crystalline forms.

[0105] The CPPs and / or the peptide barcodes disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by E. W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the CPPs and / or the peptide barcodes disclosed herein can be formulated such that an effective amount of the CPP and / or the peptide barcode is combined with a suitable carrier in order to facilitate effective administration of the CPP and / or the peptide barcode. The compositions used can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art. Examples of carriers or diluents for use with the CPPs and / or the peptide barcodes include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 100% by weight of the total of one or more of the CPPs and / or the peptide barcodes of the present technology based on the weight of the total composition including carrier or diluent.

[0106] Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The 34 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.

[0107] The CPPs and / or the peptide barcodes disclosed herein, and compositions comprising them, can be delivered to a cell either through direct contact with the cell or via a carrier means. Carrier means for delivering CPPs and / or the peptide barcodes of the present technology and compositions to cells are known in the art and include, for example, encapsulating the composition in a liposome moiety. Another means for delivery of the CPPs and / or the peptide barcodes disclosed herein and compositions disclosed herein to a cell comprises attaching the CPPs alone or in combination with the peptide barcodes to a protein or nucleic acid that is targeted for delivery to the target cell. U.S. Pat. No.6,960,648 and U.S. Application Publication Nos.20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and that allows the composition to be translocated across biological membranes. U.S. Application Publication No.20020035243 also describes compositions for transporting biological moieties across cell membranes for intracellular delivery. The CPPs and / or the peptide barcodes disclosed herein can also be incorporated into polymers, examples of which include poly (D-L lactide-co-glycolide) polymer for intracranial tumors; poly[bis(p- carboxyphenoxy) propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL); chondroitin; chitin; and chitosan.

[0108] In certain examples, CPPs, peptide barcodes, and compositions disclosed herein can be locally administered at one or more anatomical sites, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. CPPs, peptide barcodes and compositions disclosed herein can be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, or 35 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

[0109] The disclosed compositions are bioavailable and can be delivered orally. Oral compositions can be tablets, troches, pills, capsules, and the like, and can also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring can be added. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir can contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound can be incorporated into sustained-release preparations and devices.

[0110] CPPs, peptide barcodes and compositions disclosed herein, including pharmaceutically acceptable salts or prodrugs thereof, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. 36 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0111] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0112] Sterile injectable solutions are prepared by incorporating an agent disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0113] For topical administration, CPPs and / or peptide barcodes disclosed herein can be applied in as a liquid or solid. However, it will generally be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which can be a solid or a liquid. CPPs, the peptide barcodes, and compositions disclosed herein can be applied topically to a subject's skin to treat a disease. Preferably, the CPPs, peptide barcodes and conjugates thereof are applied to a tissue site in a formulation such as an ointment, cream, lotion, solution, tincture, or the like. 37 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0114] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.

[0115] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0116] Useful dosages of the CPPs and / or the peptide barcodes and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0117] The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

[0118] Also disclosed are pharmaceutical compositions that comprise a CPP, a peptide barcode or a conjugate disclosed herein in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions adapted for oral, topical or parenteral administration, comprising an amount of a compound constitute a preferred aspect. The dose administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient 38 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 over a reasonable time frame, without lethal toxicity, and preferably causing no more than an acceptable level of side effects or morbidity. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition.

[0119] Any method known to those in the art for contacting a cell, organ or tissue with one or more CPPs, alone or in combination with the peptide barcodes, or conjugates disclosed herein may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of one or more CPPs, alone or in combination with the peptide barcodes, or conjugates to a mammal, suitably a human. When used in vivo for therapy, the one or more CPPs, alone or in combination with the peptide barcodes, or conjugates described herein are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the disease state of the subject, the characteristics of the particular CPP, alone or in combination with the peptide barcode, or conjugate used, e.g., its therapeutic index, and the subject’s history.

[0120] The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of one or more CPPs, alone or in combination with the peptide barcodes, or conjugates useful in the methods may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The CPP, alone or in combination with the peptide barcode, or conjugate may be administered systemically or locally.

[0121] The one or more CPPs, alone or in combination with the peptide barcodes, or conjugates described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment or prevention of a disease or condition. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and 39 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0122] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment).

[0123] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0124] The pharmaceutical compositions having one or more CPPs, alone or in combination with the peptide barcodes, or conjugates disclosed herein can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, 40 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0125] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0126] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; 41 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0127] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

[0128] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed by iontophoresis.

[0129] A therapeutic agent can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic agent is encapsulated in a liposome while maintaining the agent’s structural integrity. One skilled in the art would appreciate that there are a variety of methods to prepare liposomes. (See Lichtenberg, et al., Methods Biochem. Anal., 33:337-462 (1988); Anselem, et al., Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems. 42 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0130] The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic agent can be embedded in the polymer matrix, while maintaining the agent’s structural integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly α-hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic / glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).

[0131] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99 / 15154 (Tracy, et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale, et al.), PCT publication WO 96 / 40073 (Zale, et al.), and PCT publication WO 00 / 38651 (Shah, et al.). U.S. Pat. Nos.5,674,534 and 5,716,644 and PCT publication WO 96 / 40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.

[0132] In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811. 43 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0133] The therapeutic compounds can also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, e.g., Chonn and Cullis, “Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in Biotechnology 6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods, 4(3):201-9 (1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery: Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995). Mizguchi, et al., Cancer Lett., 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro. Effective Dosages

[0134] Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0135] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine useful doses in humans accurately. Levels in plasma may be measured, for example, by high performance liquid chromatography. 44 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0136] Typically, an effective amount of the one or more CPPs, alone or in combination with the peptide barcodes, or conjugates disclosed herein sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example, dosages can be 1 mg / kg body weight or 10 mg / kg body weight every day, every two days or every three days or within the range of 1-10 mg / kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of the therapeutic compound ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, one or more CPP or conjugate concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0137] In some embodiments, a therapeutically effective amount of one or more CPPs, alone or in combination with the peptide barcodes, or conjugates may be defined as a concentration of inhibitor at the target tissue of 10-32to 10-6molar, e.g., approximately 10-7molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg / kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).

[0138] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments. 45 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0139] The mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human. Methods of Making the Peptides of the Present Technology

[0140] The CPPs, peptide barcodes, or conjugates described herein can be prepared in a variety of ways known to one skilled in the art of organic synthesis or variations thereon as appreciated by those skilled in the art. The CPPs, peptide barcodes, or conjugates described herein can be prepared from readily available starting materials. Optimum reaction conditions can vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art.

[0141] Variations on the CPPs, peptide barcodes, or conjugates described herein include the addition, subtraction, or movement of the various constituents as described for each peptide. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0142] The starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), Sigma (St. Louis, Mo.), Pfizer (New York, N.Y.), GlaxoSmithKline (Raleigh, N.C.), Merck (Whitehouse Station, N.J.), Johnson & Johnson (New Brunswick, N.J.), Aventis (Bridgewater, N.J.), AstraZeneca (Wilmington, Del.), Novartis (Basel, Switzerland), Wyeth (Madison, N.J.), Bristol-Myers-Squibb (New York, N.Y.), Roche (Basel, Switzerland), Lilly (Indianapolis, Ind.), Abbott (Abbott Park, Ill.), Schering Plough (Kenilworth, N.J.), or Boehringer Ingelheim 46 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 (Ingelheim, Germany), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical carriers disclosed herein can be obtained from commercial sources.

[0143] Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, i.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,1H or13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0144] The disclosed compounds can be prepared by solid phase peptide synthesis wherein the amino acid α-N-terminal is protected by an acid or base protecting group. Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation while being readily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained therein. Suitable protecting groups are 9- fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, α,α-dimethyl-3,5- dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. Other preferred side chain protecting groups are, for side 47 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 chain amino groups like lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, and adamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy-carbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl and acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan, formyl; for aspartic acid and glutamic acid, benzyl and t-butyl and for cysteine, triphenylmethyl (trityl). In the solid phase peptide synthesis method, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are those materials which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the media used. Solid supports for synthesis of α-C- terminal carboxy peptides is 4-hydroxymethylphenoxymethyl-copoly(styrene-1% divinylbenzene) or 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin available from Applied Biosystems (Foster City, Calif.). The α-C-terminal amino acid is coupled to the resin by means of N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC) or O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU), with or without 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazol-1- yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP) or bis(2-oxo-3- oxazolidinyl)phosphine chloride (BOPCl), mediated coupling for from about 1 to about 24 hours at a temperature of between 10° C. and 50° C. in a solvent such as dichloromethane or DMF. When the solid support is 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy- acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine, preferably piperidine, prior to coupling with the α-C-terminal amino acid as described above. One method for coupling to the deprotected 4 (2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer. In one example, the α-N- terminal in the amino acids of the growing peptide chain are protected with Fmoc. The removal of the Fmoc protecting group from the α-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then 48 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 introduced in about 3-fold molar excess, and the coupling is preferably carried out in DMF. The coupling agent can be O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.). At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either in successively or in a single operation. Removal of the polypeptide and deprotection can be accomplished in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising thioanisole, water, ethanedithiol and trifluoroacetic acid. In cases wherein the α-C-terminal of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide can be removed by transesterification, e.g. with methanol, followed by aminolysis or by direct transamidation. The protected peptide can be purified at this point or taken to the next step directly. The removal of the side chain protecting groups can be accomplished using the cleavage cocktail described above. The fully deprotected peptide can be purified by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (for example, Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography, e.g. on Sephadex G-25, LH-20 or countercurrent distribution; high performance liquid chromatography (HPLC), especially reverse-phase HPLC on octyl- or octadecylsilyl-silica bonded phase column packing. Methods of Use

[0145] In one aspect, the present disclosure provides a method for delivering a cargo to target tissues or target cells in a subject comprising administering to the subject an effective amount of any and all embodiments of the conjugates described herein. The target tissues or target cells may comprise hematopoietic cells, mesenchymal cells, endothelial cells or epithelial cells. In some embodiments, the target tissues or target cells comprises tissues or cells from bone marrow, colon, rectum, eye, gall bladder, kidney, mouth, larynx, esophagus, stomach, intestine, lung, testis, ovary, cervix, brain, liver, skin, spinal cord, thyroid, vagina, vulva, uterus, or muscle. 49 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0146] In one aspect, the present disclosure provides a method for quantifying macromolecules in a biological sample via high-resolution mass spectrometry comprising (a) coupling macromolecules to a barcoded cell-penetrating peptide, wherein the barcoded cell- penetrating peptide comprises at least one peptide barcode of the present technology to generate a plurality of barcoded macromolecules; (b) incubating a biological sample with the plurality of barcoded macromolecules for a specified period of time; (c) performing cell fractionation on the incubated biological sample to obtain a plurality of fractions including a nuclear fraction and a cytoplasmic fraction; (d) performing high-resolution mass spectrometry on the plurality of fractions; and (e) quantifying absolute abundance of the barcoded macromolecules in the plurality of fractions based on calibration curves generated from the barcoded cell-penetrating peptide in a reference sample. The biological sample and the reference sample may be the same sample type. In some embodiments, the reference sample comprises cell lysate from target cells or target tissues. Additionally or alternatively, in some embodiments, the target tissues or target cells comprise hematopoietic cells, mesenchymal cells, endothelial cells or epithelial cells. Examples of target tissues or target cells include tissues or cells derived from bone marrow, colon, rectum, eye, gall bladder, kidney, mouth, larynx, esophagus, stomach, intestine, lung, testis, ovary, cervix, brain, liver, skin, spinal cord, thyroid, vagina, vulva, uterus, or muscle.

[0147] In another aspect, the present disclosure provides a method for selecting a cell- penetrating peptide for quantifying macromolecules in a biological sample via high-resolution mass spectrometry comprising (a) coupling a plurality of cell-penetrating peptides to at least one peptide barcode of the present technology to generate a plurality of barcoded cell-penetrating peptides; (b) incubating each barcoded cell-penetrating peptide with a biological sample for a specified period of time; (c) performing cell fractionation on the incubated biological sample of step (b) to obtain a plurality of fractions including a nuclear fraction and a cytoplasmic fraction; (d) performing high-resolution mass spectrometry on the plurality of fractions of step (c); (e) quantifying mass spectrometry signals detected in step (d) for each barcoded cell-penetrating peptide; and (f) selecting a cell-penetrating peptide based on its mass spectrometry signals in the nuclear fraction and the cytoplasmic fraction. 50 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0148] In some embodiments, the specified period of time ranges from 10 minutes to 72 hours. Additionally or alternatively, in some embodiments, the specified period of time ranges from 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 12.5 hours, 13 hours, 13.5 hours, 14 hours, 14.5 hours, 15 hours, 15.5 hours, 16 hours, 16.5 hours, 17 hours, 17.5 hours, 18 hours, 18.5 hours, 19 hours, 19.5 hours, 20 hours, 20.5 hours, 21 hours, 21.5 hours, 22 hours, 22.5 hours, 23 hours, 23.5 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours.

[0149] In any of the preceding embodiments of the methods disclosed herein, the macromolecules comprise one or more of drugs, small molecules, nucleic acids, peptidomimetics, peptides or polypeptides. Additionally or alternatively, in some embodiments, the biological sample is obtained from a subject. The subject may be healthy or is diagnosed with or at risk for a disease or condition.

[0150] In yet another aspect, the present disclosure provides a method for tracking delivery of cargo molecules in a subject in real-time comprising (a) coupling cargo molecules to a barcoded cell-penetrating peptide, wherein the barcoded cell-penetrating peptide comprises at least one peptide barcode of the present technology to generate a plurality of barcoded cargo molecules; (b) administering the plurality of barcoded cargo molecules to the subject; (c) obtaining a biological sample from the subject at one or more time periods post administration of the barcoded cargo molecules, wherein the biological sample comprises target cells or tissues; (d) performing cell fractionation on the biological sample from the one or more time periods to obtain a plurality of fractions including nuclear fractions and cytoplasmic fractions; (e) performing high-resolution mass spectrometry on the plurality of fractions; and (f) determining real-time delivery of cargo molecules in the subject by identifying / quantifying the mass spectrometry signals of the plurality of barcoded cargo molecules in the nuclear fractions and the cytoplasmic fractions. The subject may be healthy or is diagnosed with or at risk for a disease or condition. 51 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0151] In some embodiments, the one or more time periods range from at least 1 minute to at least 72 hours post administration of the barcoded cargo molecules. Additionally or alternatively, in some embodiments, the one or more time periods range from at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, at least 10.5 hours, at least 11 hours, at least 12.5 hours, at least 13 hours, at least 13.5 hours, at least 14 hours, at least 14.5 hours, at least 15 hours, at least 15.5 hours, at least 16 hours, at least 16.5 hours, at least 17 hours, at least 17.5 hours, at least 18 hours, at least 18.5 hours, at least 19 hours, at least 19.5 hours, at least 20 hours, at least 20.5 hours, at least 21 hours, at least 21.5 hours, at least 22 hours, at least 22.5 hours, at least 23 hours, at least 23.5 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours.

[0152] Additionally or alternatively, in certain embodiments, the cargo molecules comprise one or more of drugs, small molecules, nucleic acids, peptidomimetics, peptides, or polypeptides.

[0153] In any and all embodiments of the methods disclosed herein, the cell fractionation is achieved using digitonin and sucrose density sedimentation. In any and all embodiments of the methods disclosed herein, the biological sample comprises blood, plasma, saliva, urine, serum, CSF, or biopsied tissue. Kits

[0154] In one aspect, the present disclosure provides a kit comprising one or more peptide barcodes comprising an amino acid sequence selected from any one of SEQ ID NOs: 3-98, and instructions for using the same to quantify macromolecules in a biological sample via high- resolution mass spectrometry. The kit may further comprise one or more cell-penetrating peptides and instructions for linking the one or more cell-penetrating peptides to the one or more peptide barcodes. In some embodiments, the biological sample comprises blood, plasma, saliva, urine, serum, CSF, or biopsied tissue. 52 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0155] In another aspect, the present disclosure provides a kit comprising one or more chimeric cell-penetrating polypeptides (CPPs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 101-104, and instructions for linking the same to a cargo molecule that is configured to be delivered to target tissues or target cells. The kit may further comprise one or more peptide barcodes and instructions for linking the one or more peptide barcodes to the one or more CPPs.

[0156] In yet another aspect, the present disclosure provides a kit comprising at least one CPP of the present technology, at least one peptide barcode of the present technology, and instructions for using the same to monitor delivery of macromolecules in a biological sample or a subject. In some embodiments, wherein the macromolecules comprise one or more of drugs, small molecules, nucleic acids, peptidomimetics, peptides or polypeptides. The biological sample may comprises blood, plasma, saliva, urine, serum, CSF, or biopsied tissue.

[0157] The disclosed kits can optionally include pharmaceutically acceptable carriers and / or diluents. In one embodiment, a kit includes one or more other components, adjuncts, or adjuvants as described herein. In one embodiment, a kit includes instructions or packaging materials that describe how to administer a compound or composition of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, a compound and / or agent disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, a compound and / or agent disclosed herein is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe containing a compound and / or agent disclosed herein in liquid or solution form. EXAMPLES

[0158] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. Example 1: Materials and Methods Reagents 53 4856-6309-7511.1Atty. Dkt. No.: 115872-3199

[0159] The Fmoc amino acids, OxymaPure, and Rink Amide ProTide Resin used for solid phase peptide synthesis were purchased from CEM Corporation (Charlotte, NC, USA). N,N′- diisopropylcarbodiimide (DIC), 2,2’-(Ethylenedioxy) diethanethiol (DODT), triisopropylsilane (TIPS), sodium nitrite, sodium phosphate monobasic, 4-mercaptophenylacetic acid (MPAA), trifluoroacetic acid (TFA), piperidine, dimethylformamide (DMF), dichloromethane (DCM), diethyl ether, guanidine hydrochloride (Gn^HCl), 2-mercaptoethanol, and tris(2- carboxyethyl)phosphine hydrochloride (TCEP^HCl) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Va-044) was purchased from Wako Chemicals (Richmond, VA, USA). HPLC-grade reagents including water, methanol, acetonitrile, and formic acid were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Digitonin was purchased from MilliporeSigma (Burlington, MA, USA). cOmplete protease inhibitors were obtained from Roche Diagnostics GmbH (Mannheim, Germany). Synthetic Chemistry

[0160] Peptide barcodes and CPPs were synthesized using the Liberty Blue HT12 microwave peptide synthesizer, according to the manufacturer’s instructions (CEM Corporation, Charlotte, NC).17, 180.2 M stock solutions of amino acids were made in DMF. 20% piperidine in DMF was used as the Fmoc-deprotecting solvent. 1 M OxymaPure ethyl 2-cyano-2- (hydroxyimino)acetate was used with 0.5 M N,N′-diisopropylcarbodiimide (DIC) in the carbodiimide approach to form peptide bonds, with inhibition of racemization and improved coupling efficiencies.19All the barcode sequences were designed with N-terminal alanine (A) and C-terminal arginine (R), and one tryptophan (W) in their sequences to aid in UV absorbance detection, as listed in FIG.10. In order to proceed with native chemical ligation, all the barcode peptides were initially synthesized with N-terminal cysteine (C), instead of alanine (A), using rink amide resin resulting in C-terminal amides. CPP peptides were synthesized using 2- chlorotrityl chloride (CTC) resin to prepare C-terminal hydrazide (R-NHNH2).20The synthesized peptides were cleaved off the resin using the TFA:H2O:TIPS:DODT solution (92.5:2.5:2.5:2.5), and purified by adding 10-fold volume of cold diethyl ether to the TFA 54 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 cleavage cocktail. Short barcode peptides (FIG.10) used for generating signal-response standard curves were synthesized as SpikeTides (JPT Technologies, Berlin, Germany).

[0161] Barcoded CPP libraries were generated using native chemical ligation.21All reactions were carried out in the 0.2 M phosphate buffer containing 6 M guanidine hydrochloride, pH 3 (NCL buffer). For each peptide, 3.6 mg of CPP peptide was dissolved in 0.4 ml NCL buffer, and stirred at -15°C for 15 min. Separately, 13.6 mg MPAA and 1.25 mg barcode peptide were dissolved in 0.4 ml NCL buffer, and the pH was adjusted to 6.5 by adding 1M NaOH. The pre-cooled CPP peptide solution was oxidized with 40 µL 0.5 M NaNO2 by stirring at -15°C for 15 min. MPAA and barcode mixtures were added dropwise into the tube containing CPP peptide, and the tube was then warmed to room temperature, and its pH was adjusted to 6.8-7.0 by adding 1M NaOH. After overnight incubation with stirring at room temperature, reaction mixtures were reduced by the addition of 0.4 ml of 0.1 M TCEP (NCL buffer, pH 6.0-7.0) and stirring for 20 minutes.22Cysteine desulfurization was carried out as described in.23Specifically, 2 mg of NCL-ligated barcoded-CPP was dissolved in 300 µL of NCL buffer, into which 300 uL of aqueous 0.5 M TCEP^HCl, 20 uL of 2-mercaptoethanol (10% v / v), and 20 uL of 0.1 M VA-044 were added. The pH of reaction mixture was adjusted with 1 M HCl and NaOH to neutral, and the reaction was stirred at 45°C for 45 min. All peptides were purified by high-performance liquid chromatography (HPLC) using with XBridge Peptide BEH C18 OBD Prep Column (#186008193, Waters, Milford, MA) and acetonitrile gradient 5-40% with 0.1% trifluoroacetic acid, to achieve at least 90% purity as measured by LC-MS. Cell culture

[0162] Kasumi-1, HEK293T, and HUVEC cells were obtained from American Type Culture Collection (ATCC, Manassas, Virginia, USA). Huh-7 cells were kindly provided by Dr. Hao Zhu from UT Southwestern Medical Center. All cell lines were verified by short tandem repeats (STR) analysis (Integrated Genomics Operation, Memorial Sloan Kettering Cancer Center, New York, NY, USA). Cultures were confirmed to be free of mycoplasma contamination using Lonza MycoAlert (Lonza Walkersville, Inc., Walkersville, MD, USA). Cells were cultured at a concentration of 1x10^6 cells / mL in 5% CO2 in humidified atmosphere at 37 °C, in media 55 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 consisting of 10% fetal bovine serum, 100 U / mL penicillin, and 100 ug / mL streptomycin. RPMI-1640 medium (Corning Life Science, Corning, NY, USA) was used for Kasumi-1 cells; DMEM medium (Corning Life Science, Corning, NY, USA) was used for HEK293T and Huh-7 cells; the EGM-2 Endothelial Cell Growth Medium (Lonza Inc., Morristown, NJ, USA) was used for HUVEC cells. Membrane permeabilization and cytotoxicity measurements

[0163] Membrane stability was evaluated using lactate dehydrogenase (LDH) release assay (#ab65393, AbCam, Cambridge, UK) and cell viability was measured using the CellTiter-Glo luminescent assay, according to manufacturer’s instructions (#G7571, Promega Corporation, Madison, WI, USA). Cells were counted with Countess II Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA) and 10K cells were aliquoted into each well of 96-well cell culture plates. Suspension Kasumi-1 cells were aliquoted immediately before treatment, while adherent HEK293T, Huh-7 and HUVEC cells were plated 17 hours before treatment. Cells were treated as indicated in complete media, and assayed using the TECAN Infinite M1000 Pro microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Cell fractionation

[0164] Cells were counted using the Countess II Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA) and aliquoted into 12-well cell culture plates (for arrayed treatment), 10 cm petri dishes and 25 cm2 flasks (for pooled treatment). Suspension Kasumi-1 cells (300K) were aliquoted immediately before treatment, while adherent HEK293T (175K), Huh-7 (240K) and HUVEC (280K) cells were plated 17 hours before treatment. Cells were treated as indicated in complete media, and washed once in PBS using centrifugation. For cell fractionation, a cell pellet of 2-3 million cells was resuspended in 90-150 µL of hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM NaCl, 1 mM MgCl2, 0.5 mM DTT, complete protease inhibitors) containing 0.1% digitonin, and was incubated at 25 °C for 5 min. The suspension was then centrifuged for 15 min at 3300 g at 4°C, and cytoplasmic supernatant was collected. The nuclei pellet was then resuspended in 500 µL of sucrose resuspension buffer containing 0.25 M sucrose and 10 mM MgCl2 (PBS buffer, pH 7.4). The suspension was layered onto a 500 µL 56 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 sucrose cushion buffer (0.88 M sucrose and 0.05 mM MgCl2 in PBS, pH 7.4), and centrifuged at 1200 g at 4°C for 11 min. The nuclei pellet thus obtained was subjected to nuclear lysis and protein extraction in 70-110 µL of lysis buffer (6 M guanidine hydrochloride in PBS buffer, pH 7.4), sonicated by Covaris S220 ultrasonicator (Covaris, LLC., Woburn, MA, USA) at peak power 125W, duty factor of 10%, cycle / burst of 200, and duration of 360 sec. The protein content in both nuclear and cytoplasmic fractions was quantified using Pierce BCA Protein Assay Kit, according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). Purified samples were stored at -80 °C. Proteomics sample preparation

[0165] Purified protein extracts were digested with sequencing grade modified trypsin protease (Promega Corporation, Madison, WI, USA), with a protein-to-trypsin mass ratio of 10:1. The digestion mixture was incubated at 37 °C overnight. Digestion was halted by addition of formic acid to 3.36% v / v, and 650 fmol of the Pierce Retention Time Calibration Mixture (PRTC) was added into each sample as internal standards. Tryptic peptides were purified using solid phase extraction with BioPureSPN MIDI columns (#HEM S18V, Nest Group, Southborough, MA, USA) according to manufacturer’s protocol. Briefly, the spin column was washed with 200 µL methanol and activated with 200 µL acetonitrile, then equilibrated twice with 200 µL 0.1% formic acid in water. Samples were loaded onto equilibrated columns, and then washed twice with 200 µL 0.1% formic acid in water. Peptides were eluted with 60% acetonitrile containing 0.1% formic acid, and lyophilized by vacuum centrifugation. For analysis, purified peptides were resuspended in 0.1% formic acid in water. Calibration curves of barcoded CPPs were established by adding variable amounts of synthetic peptides into cell lysates, followed by their digestion and purification as described above. Nanoscale liquid chromatography and nanoelectrospray ionization mass spectrometry

[0166] All nanoscale liquid chromatography was performed using the Eksigent nanoLC425 chromatography system (Sciex, Framingham, MA, USA). The 50 cm uPAC pillar array reverse phase column (#COL-NANO050G1B, ThermoFisher Scientific, San Jose, CA, USA) was used for the LC-MS signal-response study of the pilot 96 peptide barcodes. The capillary reverse 57 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 phase columns were used for cellular delivery quantitation. These capillary columns were fabricated by pressure filling the stationary phase into silica capillaries fritted with K-silicate, as previously described.24Samples were resolved with constant flowrate of 300 nL / min using a 5- 40% linear gradient of acetonitrile in water (both with 0.1% v / v / formic acid) over 90 minutes. Ionization was accomplished using laser-fabricated emitters with terminal opening diameter of 2- 3 µm, made from 50 µm silica capillaries, and connected to the outlet of the reversed phase column using a metal union that also served as the electrospray current electrode, as described.25Eluting peptides were transferred to an Orbitrap Fusion mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA) via the DPV-566 PicoView nano-electrospray ion source (New Objective, Woburn, MA, USA).

[0167] For data dependent acquisition (DDA), the full-scan spectra were acquired in positive ion mode at a fixed resolution of 120,000 and a mass range of 300-1200 m / z. Fragmentation spectra were acquired at a resolution of 7,500, and the precursor ions were isolated using 1.6 m / z width and fragmented with a fixed higher-energy collisional dissociation (HCD) collision energy of 30%.

[0168] For parallel reaction monitoring (PRM) scans, full-scan spectra were acquired in positive ion mode at a mass range of 300-1200 m / z and 120,000 resolution. Precursor ions were isolated in the quadrupole using 1.2 m / z windows and fragmented by HCD with normalized collision energy 30% (stepped collision energy + / -5%), before analysis of fragment ions in the Orbitrap at 7,500 resolution. The PRM method was scheduled using 10-min acquisition windows set for each barcode based on the DDA retention time results.26

[0169] For selected ion monitoring (SIM) scans, the precursor ions were isolated in the quadrupole using 0.7 m / z window and were detected in the Orbitrap at 240,000 resolution. The maximum injection time mode was set as dynamic, with six minimum points across the peak. The SIM method was scheduled using 10-min acquisition windows set for each barcode based on the DDA retention time results.

[0170] The mass spectra obtained from all methods were analyzed using Skyline27(version 21.2.0.425). The target list was first established in Skyline, then the MS raw data files were 58 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 imported to extract the target chromatographs. DDA MS1 spectra were filtered with a resolving power of 120,000 at m / z 400. SIM MS1 spectra were filtered with a resolving power of 240,000 at m / z 200. PRM MS2 spectra were filtered using 60,000 resolving power at m / z 200, and at least 3 fragments per precursor are required for valid quantitation. For the LC-MS signal- response study, the MS1 and MS2 ion intensities of each barcode precursor in all serially diluted samples were extracted and were linearly fitted to obtain a signal-response function for each barcode. For the absolute quantitation of digested cell fractions, MS2 ion intensities of all barcodes were exported and normalized to the ion intensity of PRTC ionization standards. A baseline sample (untreated cell lysate) was measured, and any ion intensities of the cell fractions that were lower or comparable to the baseline signal were considered non-detectable. Only valid signals from the cell fractions were calculated using the calibration curves to obtain the final delivery quantities.

[0171] Numerical and statistical analyses were performed using Origin Pro (OriginLab Corporation, Northampton, MA). BarcodeBabel

[0172] BarcodeBabel is a Python algorithm to generate libraries of peptide barcodes with user-defined ranges of features for optimal detectability by nanoscale liquid chromatography tandem mass spectrometry. User-selected rules include user-defined m / z range, absence of homopolymers, specific hydrophobicity range, enzyme cleavage sites, residue frequencies, and library size. In addition, users can specify reference proteome to remove any naturally occurring interfering sequences. The default enzyme for barcode cleavage is trypsin. Specific residues are programmed to be avoided: lysine (K), arginine (R), and histidine (H) were omitted to prevent trypsin cleavage within the barcode sequences; methionine (M) and cysteine (C) were omitted to avoid oxidation and crosslinking events; proline (P) was omitted as it may skew fragmentation; glutamine (Q) and asparagine (N) were avoided due to their propensity for deamidation; isoleucine (I) was omitted because it cannot be distinguished from leucine (L) by conventional mass spectrometry. Peptide properties were calculated at default pH of 3 (for conventional positive ion mode electrospray). Default m / z range was set to 550-850 to fall within the optimal 59 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 detection window of high-field Orbitrap mass analyzers. Default hydrophobicity range (-0.5 to 2.5) is specified to allow for consistent peptide elution in the middle of conventional reverse phase chromatography gradients. Users can also list specific reference proteomes, as well as common contaminants to avoid sequence overlap. BarcodeBabel is implemented open-source via https: / / github.com / kentsisresearchgroup / BarcodeBabel. PeptideBabel

[0173] PeptideBabel is Python algorithm for the generation of novel bioactive peptide sequences using Metropolis-Hastings sampling. This algorithm generates novel peptides by exploring the sequence space around a set of seed sequences using Markov chain Monte Carlo sampling (Metropolis-Hastings) with permutation of seed sequences mapped to a density function based on physicochemical features or k-mer sequence complexity, as specified by users. This allows efficient generation of hundreds to millions of peptide sequences for subsequent empiric validation, tailored to application based on input peptides. User uploads list of references peptides (seed library). PeptideBabel then defines properties of the seed library, including sequence length, hydrophobicity (windowed Kyte-Doolittle scale), isoelectric point (Bjellqvist), secondary structure propensity (fraction helical, turn-like, and sheet-like based on residue composition using the Garnier-Osguthorpe-Robson method). Sampling algorithm permutes peptide sequences (substitutions, insertions, deletions) at user defined sequence length constraints and sampling density, and implements a random-walk Metropolis-Hastings, favoring steps moving up the density function at user-defined probability to generate new sequences or down the density function to sample similar sequences. PeptideBabel is implemented open- source via https: / / github.com / kentsisresearchgroup / PeptideBabel. Example 2: Analysis of Barcode and CPP peptides

[0174] Mass spectrometry proteomics enables the detection and quantitation of specific macromolecules based on tandem fragmentation and high-accuracy measurements of their mass and charge. This enables the resolution of unique polypeptide sequences, differing by as little as a single amino acid. To enable the generation of libraries of unique peptide barcodes for studies of engineered macromolecules in complex biological samples, BarcodeBabel, a python script 60 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 and corresponding Jupyter notebook, was developed which computes arbitrary numbers of unique amino acid peptide sequences, flanked by specific enzyme cleavage sites for their release in complex biological samples, e.g. trypsin (FIG.1A). The script can exclude user-defined internal amino acids which may interfere with correct enzymatic cleavage and consistent mass spectrometric detection or fragmentation, i.e. lysine, arginine, histidine, cysteine, methionine, proline, and isoleucine. Users may also specify frequencies of amino acids to generate libraries with specific features, such as inclusion of tryptophan residue for optical measurements, as well as specific ranges of m / z values and amino acid lengths. The script also implements a hydrophobicity estimator based on the Kyte-Doolittle scale for optimal reverse phase separations that accompany high-resolution mass spectrometry measurements. To calibrate this parameter, 175,579 unique tryptic peptides of length from 5 to 15 residues were analyzed,28and found that peptides with hydrophobicity scores of -0.5 to 2.5 exhibit monotonically variable retention times in reverse phase chromatography most often coupled with modern high-resolution mass spectrometry instruments, which was implemented as the default values for BarcodeBabel (FIGs.9A-9D). Finally, BarcodeBabel is configured with a user-specified reference proteome to ensure that the designed barcode sequences do not match any naturally occurring or common contaminant proteins.

[0175] To generate new sequences to test for cell penetration properties, potential peptide candidates from viral sequences (FIG.11A) were catalogued. ViralZone (https: / / viralzone.expasy.org) human pathogens were searched for presence of known penetration domains using UniProt annotations and literature search for viral binding and entry studies for each virus. Fusion peptides on enveloped viruses are often hydrophobic, rich in Gly residues at the N-terminus. Fusion peptides are theorized to insert into the target membrane and destabilize the bilayer, although a pitfall of synthetic viral fusion peptides is that they are less effective in accelerating membrane fusion than full domains or viruses, likely due to the need for cooperating domains. Non-enveloped viruses consist of one major capsid protein and occasionally additional minor capsid proteins, and enveloped virions also contain nucleocapsid proteins. Some physicochemical properties for candidate peptide sequences, in comparison to previously catalogued peptides from CPPiste2.06are shown in FIGS.11B-11C. Some observed trends 61 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 include increased hydrophobicity of envelope fusion peptides which is related to membrane interactions, and higher isoelectric point of capsid peptides with presence of cationic nuclear localization sequences.

[0176] To test BarcodeBabel, a library of 96 unique barcode sequences was designed, synthesized using solid phase synthesis, and purified peptides were serially diluted in whole-cell extracts of human OCI-AML2 cells. The abundance of peptides in whole-cell proteomes was quantified using selected ion monitoring (SIM), data-dependent acquisition (DDA), and parallel reaction monitoring (PRM), with limits of quantitation determined using Skyline. It was found that PRM exhibited superior limits of quantitation (LOQ), as compared to SIM and DDA (mean LOQ of 75 versus 550 and 280 amol, respectively; FIGs.1B-1D). It was also found that PRM exhibited more uniform signal-response for the quantitation of barcode peptide abundance, as compared to SIM and DDA (mean 0.67 ± 0.22 versus 0.60 ± 0.34 and 0.80 ± 0.30 intensity / amol, respectively; FIG.1E). Thus, BarcodeBabel permits the construction of libraries of specific peptide barcodes, which can be quantitatively deconvoluted using high-resolution mass spectrometry.

[0177] Pioneering studies of first-generation of CPPs and PTDs for macromolecular delivery used naturally inspired peptides derived from TAT and penetratin.29, 30Since then, a variety of cationic and amphipathic CPPs have been identified experimentally, as most recently catalogued in the CPPsite 2.0 database.31, 32Comparative studies of specific CPPs have identified several key propensities, such as the optimal number of eight guanidine side chains for cationic polyarginine CPPs.33However, the development of explicit structure-activity relationships for efficient, selective and safe CPPs and PTDs has been challenging, at least in part due to the diversity and complexity of CPPs and their membrane penetration and cellular internalization mechanisms.

[0178] It was reasoned that the mechanisms of CPP membrane penetration and cellular internalization ultimately can be learned from large-scale structure-function studies. To enable the generation of libraries of candidate CPPs for high-throughput studies, a Monte Carlo sampling algorithm PeptideBabel was implemented. PeptideBabel uses the Metropolis-Hastings 62 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 algorithm to introduce random changes in the amino acid composition of seed peptide sequences, followed by their acceptance or rejection based on user-specified density functions of either the physicochemical properties of peptides or their k-mer sequence complexity (FIG.2). Current version of PeptideBabel includes 1552 seed sequences of putative membrane penetration domains from diverse viral pathogens,34as well as curated members from CPPsite 2.0 (FIG. 11A-11C, data not shown). PeptideBabel’s physicochemical scoring function is based on the linear combination of peptide length, estimated isoelectric point, hydrophobicity (Kyte- Doolittle), and secondary structure propensity (Garnier-Osguthorpe-Robson). Alternatively, PeptideBabel can sample peptide sequences based on their sequence complexity, as measured using k-mer scoring.35The Monte Carlo sampling and design can be executed to generate unique sequences with physicochemical or complexity properties that are either similar to the seed sequences, or alternatively, those that are of increasing diversity from the seed sequences. Convergence of sampling can be assessed using multiple independent calculations, starting from different initial conditions.36Thus, PeptideBabel is expected to permit the construction of diverse libraries of candidate bioactive peptides.

[0179] As proof-of-concept of this strategy, a small library of barcoded CPPs was designed, including representative cationic and amphipathic CPPs, as well as novel chimeric CPPs, and their anionic negative controls that do not transit across anionic mammalian cell membranes (FIG.12). Unique barcodes with diverse physicochemical properties, as estimated by their m / z, net charge, and hydrophobicity values were chosen (FIGs.13A-13D). Barcode peptides and CPPs were synthesized using solid phase synthesis, and assembled using native chemical ligation.22Because native chemical ligation involves cysteines that can undergo chemical reactions in cells, desulfurization was used to convert them into alanines.23This allowed barcoded CPPs up to 42-amino acids in length to be generated with >90% purity, as confirmed using LC-MS (FIG.14).

[0180] CPPs can disrupt cellular membranes, causing both cytotoxicity and artifactual internalization due to cell death. Therefore, the effects of individual barcoded CPPs on cell viability was measured, as assessed with cellular ATP content, and membrane stability with 63 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 assays of LDH release using a panel of four biologically diverse cell lines: hematopoietic Kasumi-1 cells, mesenchymal HEK293T cells, endothelial HUVEC cells, and epithelial Huh-7 cells (FIGs.3A-3F, FIG.15, and FIG.16). It was found that TAT-[barcode64] CPP showed no measurable cytotoxicity up to 15 µM either at 3 or 24 hours of exposure on Kasumi-1 cells, and up to 100 µM across the other three cell lines tested. In contrast, 3-hour incubation of chimeric KLAL-TAT-[barcode82] CPP promoted LDH release and impaired cell viability with an IC50 of 1.4 and 2.3 µM, respectively in HUVEC cells, and 34 and 10 µM, respectively in HEK293T cells, with the other two cell lines having intermediate values (FIGs.3A-3F, FIG.15, and FIG. 16). It was found that Kasumi-1 cells were most sensitive to various CPPs, whereas Huh-7 and HEK293T cells were largely insensitive. Importantly, all barcoded CPPs had negligible cytotoxicity and membrane disruption at 1 µM, thereby establishing this dose for cell delivery studies in vitro (FIGs.6A-6C, FIGs.7A-7D, FIGs.21A-21F, FIGs.22A-22G, and FIGs.23A- 23D). Though many prior studies have used 10-100 µM CPP cell treatments,37this lower concentration was chosen in order to avoid any potential confounding effects from membrane disruption and cytotoxicity.

[0181] To assess membrane penetration and subcellular delivery of various CPPs, it was first sought to establish a robust cellular fractionation method to measure nuclear versus cytoplasmic accumulation of barcoded CPPs. It was found that plasma membrane extraction using 0.1% digitonin, followed by sucrose density sedimentation produced specific separation of nuclear versus cytoplasmic compartments, as validated by Western immunoblotting against Lamin B1, Histone H3 and GAPDH, respectively (FIGs.4A-4B, FIG.17, FIGs.18A-18C, and FIG.19). A quantitative procedure was then established for measuring the absolute abundance of specific barcoded CPPs using cell fractionation, combined with targeted PRM mass spectrometry, with variation in ionization efficiency controlled by normalization to synthetic PRTC peptides (FIG. 5A-5C). FIG.5B shows signal-response function for representative barcode B91.

[0182] Using this approach, the potential contribution of barcode peptides was determined on the cellular penetration activity of chimeric TAT-P-Ebola CPP, chosen because it contains both cationic and amphipathic CPP components (FIGs.6A-6C). Library of TAT-P-Ebola 64 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 GAAIGLAWIPYFGPAAYPRKKRRQRRR-[barcode] CPPs containing 8 diverse peptide barcodes showed no significant variation in their membrane destabilization or cytotoxicity, as measured by LDH release and ATP content, respectively (mean IC50 = 22 ± 6.0 and 12 ± 2.2 mM, respectively; FIGs.6A-B). In contrast, TAT-P-Ebola variants with AFSVDAETLWR B82 and AGLDELAAFGWR B91 barcodes used at non-toxic 1 µM concentrations exhibited significantly enhanced cytoplasmic accumulation, as compared to B55, B64, B81, B107, B108, and B121 barcodes, which were nearly exclusively nuclear (mean cytoplasmic abundance = 22 and 9.9 millions of molecules / cell, two-tailed unpaired Student’s t-test p = 0.018 and 0.022, respectively; FIG.6C). These results indicate that peptide barcoding can be used both to quantitatively measure and to modulate biologic properties of CPPs.

[0183] The nuclear and cytoplasmic delivery of barcoded CPPs in our panel of cell lines was then investigated, tested at 1 µM concentrations with their negligible cytotoxicity and membrane disruption (FIGs.3A-3F). As a negative control, the anionic version of TAT, termed badTAT, in which key arginine residues have been replaced with glutamates was used (YGEKKEEQRRR- [barcode55]), preventing its membrane translocation which consistently led to lack of measurable accumulation of badTAT either in cytoplasmic or nuclear fractions (FIG.7A). Nuclear accumulation of TAT-[barcode64] and P14-[barcode91] CPPs was detected, which is consistent with prior studies but at relatively low levels, due to the low 1 µM concentration of treatment, in order to be more relevant for future therapy development, and to minimize the confounding effects of cytotoxicity; TAT and P14 CPPs are used at >10 µM concentrations in many prior studies.14, 38

[0184] Notably, the novel chimeric CPPs TAT-P-Ebola-[barcode121], TAT-G-EBV- [barcode107], and RLAL-TAT-[barcode108] exhibited significantly higher nuclear accumulation (mean nuclear abundance = 403, 251 and 196 millions of molecules / cell, two-tailed unpaired Student’s t-test p < 0.0001 versus TAT-[barcode64]; FIG.7A). In addition, TAT-P-Ebola- [barcode121] and RLAL-TAT-[barcode108], but not TAT-G-EBV-[barcode107] exhibited time- dependent increase in nuclear accumulation after 24 hours of cell exposure (FIG.7B). Interestingly, KLAL-TAT-[barcode82] also exhibited significant time-dependent cytoplasmic 65 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 accumulation, as compared to other barcoded CPPs (mean cytoplasmic abundance = 27 and 42 millions of molecules / cell at 3 and 24 hours, two-tailed unpaired Student’s t=test p = 0.0001 and 0.0004 versus TAT-[barcode64], respectively; FIGs.7A-7B). This effect may be potentiated by the specific contribution of the AFSVDAETLWR B82 barcode to apparent CPP activity (FIG.6C).

[0185] Importantly, it was found that the activity of novel barcoded CPPs was cell type specific. For example, KLAL-TAT-[barcode82] showed increased cytoplasmic accumulation in mesenchymal HEK293T, epithelial Huh-7, and hematopoietic Kasumi-1 cells, but not in endothelial HUVEC cells (mean cytoplasmic abundance = 31, 27 and 15 versus 0 millions of molecules / cell, respectively; FIG.7C). In contrast, TAT-P-Ebola-[barcode121] exhibited increased nuclear accumulation in Huh-7 and HUVEC cells, as compared to HEK293T and Kasumi-1 cells (mean nuclear abundance = 420 and 291 versus 112 and 30.5 millions of molecules / cell, respectively; FIG.7D). Thus, the combination of peptide barcoding and de novo CPP design can be used to discover CPPs with improved cellular penetration activities and reduced toxicities (FIG.8). In addition, comparative studies can reveal time-dependent and cell type-specific differences in activity, thereby identifying potential targets for mechanistic studies.

[0186] Nucleic acid barcoding has become a highly enabling technology for diverse high- throughput biological studies, such as binding studies using diversity-oriented synthesis and DNA barcoding, peptide and protein engineering using RNA display and RNA barcoding, among others.39Recently, Egloff et al used genetically encoded peptide barcodes, as deconvoluted by mass spectrometry proteomics, for measuring binding affinities of engineered proteins.15Peptide barcoding is particularly compelling for the engineering, screening, and other studies of biological molecules, because of their homogeneous biochemical properties, superior stability and information content, in contrast to mixed macromolecules with nucleic acid barcodes.16

[0187] Here an open-source algorithm was developed, BarcodeBabel, designed for the construction of libraries of unique peptide barcodes with optimal properties for high-throughput mass spectrometry proteomics studies. This enables the design of unique sequences not present 66 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 in canonical biological proteomes, e.g., human tissues, with optimal ionization, separation, and fragmentation properties, as empirically validated using a library of 96 designed peptide barcodes.

[0188] BarcodeBabel is accompanied by an open-source algorithm PeptideBabel, which implements Monte Carlo sampling of user-defined seed sequences to generate novel libraries of peptides with diverse physicochemical and sequence complexity properties. This approach was tested empirically using a small library of barcoded cell penetration peptides (CPPs). It was found that peptide barcoding can be used to quantitatively measure cell penetration and subcellular distribution of CPPs, as validated using known CPPs and their inactive negative controls. This work also implemented a targeted mass spectrometry method, suitable for quantitative studies of absolute molecular abundance of peptide barcodes in complex biological samples.

[0189] Using this proof-of-concept study, novel CPPs were developed with improved nuclear and cytoplasmic delivery exceeding hundreds of millions of molecules per human cell, with distinct cell type specific activities, while maintaining minimal membrane disruption and negligible toxicity in vitro. Improved nuclear and cytoplasmic delivery of novel chimeric CPPs such as TAT-P-Ebola and KLAL-TAT was observed. Interestingly, it was found that in some cases, the specific barcode modulated the apparent CPP activity, suggesting that peptide barcodes may themselves be incorporated into the design of bioactive molecules. This also indicates that high-throughput screens should utilize multiple independent barcodes in order to discern specific biological activities, as is practiced with nucleic acid barcoding and other high- throughput technologies.

[0190] It is anticipated BarcodeBabel and PeptideBabel should be useful for diverse screening, design, and analytical studies. For example, this approach may be used to design novel protein binders and quantify their binding affinities and kinetics using libraries of purified barcoded proteins in vitro.15, 40Given the high sensitivity and resolving power of modern mass spectrometers, similar screens may also be performed with libraries of barcoded CPPs injected intravenously, and quantified using proteomics of specific tissues and organs in vivo. 67 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 BarcodeBabel and PeptideBabel enable the construction of barcoded libraries of peptidic macromolecules with varied biological activities, and thus should be useful for a wide variety of molecular evolution and screening applications. EQUIVALENTS

[0191] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0192] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0193] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group 68 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0194] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. 69 4856-6309-7511.1Atty. Dkt. No.: 115872-3199 REFERENCES (1) Wang, L.; Wang, N.; Zhang, W.; Cheng, X.; Yan, Z.; Shao, G.; Wang, X.; Wang, R.; Fu, C. Therapeutic peptides: Current applications and future directions. Signal Transduction and Targeted Therapy 2022, 7 (1), 48. (2) Lu, H.; Zhou, Q.; He, J.; Jiang, Z.; Peng, C.; Tong, R.; Shi, J. Recent advances in the development of protein–protein interactions modulators: mechanisms and clinical trials. Signal transduction and targeted therapy 2020, 5 (1), 213. (3) Muttenthaler, M.; King, G. F.; Adams, D. J.; Alewood, P. F. Trends in peptide drug discovery. Nature reviews Drug discovery 2021, 20 (4), 309-325. (4) Ebrahimi, S. B.; Samanta, D. 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Claims

Atty. Dkt. No.: 115872-3199 WHAT IS CLAIMED IS:

1. A chimeric cell-penetrating polypeptide (CPP) comprising an amino acid sequence selected from the group consisting of GAAIGLAWIPYFGPAAYPRKKRRQRRR (SEQ ID NO: 101), IYNGWYAYGRKKRRQRRR (SEQ ID NO: 102), KLALKLALKALKAALKLAGCYGRKKRRQRRR (SEQ ID NO: 103), and RLALRLALRALRAALRLAGCYGRKKRRQRRR (SEQ ID NO: 104).

2. A peptide barcode comprising an amino acid sequence selected from any one of SEQ ID NOs: 3-98, optionally wherein the peptide barcode is configured to be detectable by high-resolution mass spectrometry.

3. The CPP of claim 1, wherein the CPP is linked to a peptide barcode.

4. The CPP of claim 3, wherein the peptide barcode comprises an amino acid sequence selected from any one of SEQ ID NOs: 3-98.

5. The CPP of claim 3 or 4, wherein the peptide barcode is linked to the N-terminus of the CPP, the C-terminus of the CPP, or a side chain of an amino acid residue of the CPP.

6. A conjugate comprising the CPP of any one of claims 1 or 3-5 and a cargo moiety.

7. The conjugate of claim 6, wherein the cargo moiety is coupled to the N-terminus of the CPP, the C-terminus of the CPP, or a side chain of an amino acid residue of the CPP.

8. The conjugate of claim 6 or 7, wherein the CPP is located between the peptide barcode and the cargo moiety.

9. The conjugate of any one of claims 6-8, wherein the cargo moiety comprise a detectable moiety, a therapeutic moiety and / or a targeting moiety.

10. The conjugate of any one of claims 6-8, wherein the therapeutic moiety comprises a small molecule drug, a nucleic acid, a peptidomimetic, an enzyme, an antibody, a therapeutic protein, a receptor agonist, a receptor antagonist, an anticancer agent, an -75--6309-7511.1Atty. Dkt. No.: 115872-3199 antiviral agent, an antimicrobial agent, an anti-inflammatory agent, a protein inhibitor, an immunosuppressive agent, an anesthetic, or any combination thereof.

11. A method for delivering a cargo to target tissues or target cells in a subject comprising administering to the subject an effective amount of the conjugate of any one of claims 6-10.

12. The method of claim 11, wherein the target tissues or target cells comprise hematopoietic cells, mesenchymal cells, endothelial cells or epithelial cells.

13. The method of claim 11 or 12, wherein the target tissues or target cells comprises tissues or cells from bone marrow, colon, rectum, eye, gall bladder, kidney, mouth, larynx, esophagus, stomach, intestine, lung, testis, ovary, cervix, brain, liver, skin, spinal cord, thyroid, vagina, vulva, uterus, or muscle.

14. A kit comprising one or more peptide barcodes comprising an amino acid sequence selected from any one of SEQ ID NOs: 3-98, and instructions for using the same to quantify macromolecules in a biological sample via high-resolution mass spectrometry.

15. The kit of claim 14, further comprising one or more cell-penetrating peptides and instructions for linking the one or more cell-penetrating peptides to the one or more peptide barcodes.

16. The kit of claim 14 or 15, wherein the biological sample comprises blood, plasma, saliva, urine, serum, CSF, or biopsied tissue.

17. A kit comprising one or more chimeric cell-penetrating polypeptides (CPPs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 101-104, and instructions for linking the same to a cargo molecule that is configured to be delivered to target tissues or target cells.

18. The kit of claim 17, further comprising one or more peptide barcodes and instructions for linking the one or more peptide barcodes to the one or more CPPs. -76--6309-7511.1Atty. Dkt. No.: 115872-3199 19. A kit comprising at least one CPP of claim 1, at least one peptide barcode of claim 2, and instructions for using the same to monitor delivery of macromolecules in a biological sample or a subject.

20. The kit of claim 19, wherein the biological sample comprises blood, plasma, saliva, urine, serum, CSF, or biopsied tissue.

21. The kit of any one of claims 14-20, wherein the macromolecules comprise one or more of drugs, small molecules, nucleic acids, peptidomimetics, peptides or polypeptides.

22. A method for quantifying macromolecules in a biological sample via high-resolution mass spectrometry comprising a. coupling macromolecules to a barcoded cell-penetrating peptide, wherein the barcoded cell-penetrating peptide comprises at least one peptide barcode of claim 2 to generate a plurality of barcoded macromolecules; b. incubating a biological sample with the plurality of barcoded macromolecules for a specified period of time; c. performing cell fractionation on the incubated biological sample to obtain a plurality of fractions including a nuclear fraction and a cytoplasmic fraction; d. performing high-resolution mass spectrometry on the plurality of fractions; and e. quantifying absolute abundance of the barcoded macromolecules in the plurality of fractions based on calibration curves generated from the barcoded cell- penetrating peptide in a reference sample.

23. The method of claim 22, wherein the biological sample and the reference sample are the same sample type.

24. The method of claim 22 or 23, wherein the reference sample comprises cell lysate from target cells or target tissues. -77--6309-7511.1Atty. Dkt. No.: 115872-3199 25. The method of claim 24, wherein the target tissues or target cells comprise hematopoietic cells, mesenchymal cells, endothelial cells or epithelial cells.

26. The method of claim 24 or 25, wherein the target tissues or target cells comprises tissues or cells from bone marrow, colon, rectum, eye, gall bladder, kidney, mouth, larynx, esophagus, stomach, intestine, lung, testis, ovary, cervix, brain, liver, skin, spinal cord, thyroid, vagina, vulva, uterus, or muscle.

27. A method for selecting a cell-penetrating peptide for quantifying macromolecules in a biological sample via high-resolution mass spectrometry comprising a. coupling a plurality of cell-penetrating peptides to at least one peptide barcode of claim 2 to generate a plurality of barcoded cell-penetrating peptides; b. incubating each barcoded cell-penetrating peptide with a biological sample for a specified period of time; c. performing cell fractionation on the incubated biological sample of step (b) to obtain a plurality of fractions including a nuclear fraction and a cytoplasmic fraction; d. performing high-resolution mass spectrometry on the plurality of fractions of step (c); e. quantifying mass spectrometry signals detected in step (d) for each barcoded cell- penetrating peptide; and f. selecting a cell-penetrating peptide based on its mass spectrometry signals in the nuclear fraction and the cytoplasmic fraction.

28. The method of any one of claims 22-27, wherein the specified period of time ranges from 10 minutes to 72 hours.

29. The method of any one of claims 22-28, wherein the macromolecules comprise one or more of drugs, small molecules, nucleic acids, peptidomimetics, peptides or polypeptides. -78--6309-7511.1Atty. Dkt. No.: 115872-3199 30. The method of any one of claims 22-29, wherein the biological sample is obtained from a subject.

31. A method for tracking delivery of cargo molecules in a subject in real-time comprising a. coupling cargo molecules to a barcoded cell-penetrating peptide, wherein the barcoded cell-penetrating peptide comprises at least one peptide barcode of claim 2 to generate a plurality of barcoded cargo molecules; b. administering the plurality of barcoded cargo molecules to the subject; c. obtaining a biological sample from the subject at one or more time periods post administration of the barcoded cargo molecules, wherein the biological sample comprises target cells or tissues; d. performing cell fractionation on the biological sample from the one or more time periods to obtain a plurality of fractions including nuclear fractions and cytoplasmic fractions; e. performing high-resolution mass spectrometry on the plurality of fractions; and f. determining real-time delivery of cargo molecules in the subject by identifying / quantifying the mass spectrometry signals of the plurality of barcoded cargo molecules in the nuclear fractions and the cytoplasmic fractions.

32. The method of claim 31, wherein the one or more time periods range from 1 minute to at least 72 hours post administration of the barcoded cargo molecules.

33. The method of claim 31 or 32, wherein the cargo molecules comprise one or more of drugs, small molecules, nucleic acids, peptidomimetics, peptides, or polypeptides.

34. The method of any one of claims 22-33, wherein the cell fractionation is achieved using digitonin and sucrose density sedimentation.

35. The method of any one of claims 22-34, wherein the biological sample comprises blood, plasma, saliva, urine, serum, CSF, or biopsied tissue. -79--6309-7511.1Atty. Dkt. No.: 115872-3199 36. The method of any one of claims 30-35, wherein the subject is healthy or is diagnosed with or at risk for a disease or condition. -80--6309-7511.1

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