CELL PENETRATING PROTEIN ADAPTOR MOLECULES AND THEIR APPLICATION IN RESEARCH AND MEDICINE

Abstract

Cell penetrating peptides (CPPs) are established as a strategy to move cargoes into the interior of eukaryotic cells by engaging import machinery on the cell surface. In most cases the CPP is covalently linked to the cargo; it is common to express cargo proteins with a CPP extension. In some experiments a non-specific interactions (e.g., hydrophobic interactions) have been used to form CPP-cargo complexes, but this has numerous drawbacks. Transport is less efficient and the lack of specificity means that other macromolecules in the medium will also be internalized. This application describes the use of specific CPP labeled adaptor proteins that can be used to move a wide variety of cargoes into the cell interior. The prototype adaptor protein is calmodulin; it is small, stable and easily produced, and binds short (17 amino acid) targets with high affinity in the presence of calcium. A cargo produced with a calmodulin binding tag can be internalized efficiently by CPP tagged calmodulin. Because calcium in the cell interior is typically kept low, CPP-calmodulin releases cargoes inside the cell. The calmodulin binding tag can be used to efficiently purify target proteins by affinity chromatography.

Description

BRIEF DESCRIPTION OF FIGURES

The patent or application file contains at least one color figure. Copies of this patent or patent application publication with color drawing(s) will be provided upon request and payment of the necessary fee.

FIG. 1 shows a scheme for the uptake of payloads tagged with a cell penetrating peptide (CPP) by cells. FIG. 1-A shows CPP tagged payload (3) is in medium outside cell membrane (2); internal compartments include nuclei, mitochondria, and endoplasmic reticulum. FIG. 1-B shows CPP tagged payload binds to specific sites on the cell membrane. FIG. 1-C shows binding induces invagination promoted uptake machinery in membrane. FIG. 1-D shows payload distributed in endosomes formed by invagination, cytoplasm, and internal compartments.

FIG. 2 shows a scheme for the adaptor mediated uptake of payload by cells; association can be mediated by protein-protein or protein-ligand interactions. FIG. 2-A shows the association mediated by protein-protein or protein-ligand interactions of CPP tagged adaptor and cargo with adaptor ligand into binary complex. FIG. 2-B shows binding of binary complex to the cell membrane. FIG. 2-C shows internalization. FIG. 2-D shows dissociation and redistribution to internal compartments.

FIG. 3 shows the basic FRET (fluorescence resonance energy transfer) experiment to detect protein-protein interactions.

FIG. 4 shows ribbon representations of the three dimensional structure of calmodulin, used here as an adaptor. FIG. 4-A shows the structure of Ca²⁺-CaM bound to a canonical target peptide in the center of the molecule and FIG. 4-B shows the structure in the dumbbell-shaped conformation in the absence of target. The central helix breaks during recognition and binding, allowing calmodulin to wrap around the target. Ca²⁺ are shown as small speres; the protein is less ordered in the absence of Ca²⁺ (not shown). Free N and C termini are visible.

FIG. 5 shows amino acid sequence alignments of human calmodulin 1 (CALM) (SEQ ID NO: 1) and four ‘calmodulin like proteins:’ TNNC2 (SEQ ID NO: 2), CETN1 (SEQ ID NO: 3), CALLS (SEQ ID NO: 5), and CALL4 (SEQ ID NO: 4), homologous calcium biosensors.

FIG. 6 shows the amino acid sequence of the synthetic CPP adaptor protein, TAT-CaM, a CPP tagged calmodulin (SEQ ID NO: 6). The short CPP binding sequence is located directly behind the NOT1 site.

FIG. 7 shows confocal microscopy images demonstrating the uptake of a fluorescence labeled enzyme (neuronal nitric oxide synthase (nNOS)) mediated by a CPP linked calmodulin adaptor 3 hours after uptake by BHO cells. Nucleus stained blue; labeled nNOS is stained yellow with DiLight 540. FIG. 7-A shows nNOS added without CPP adaptor. Background still shows stained nNOS after washing with media. Some nNOS adheres to the cell surface; 3D cross sections show no nNOS inside cells. FIG. 7-B shows nNOS in the presence of TAT-CaM. A huge amount of nNOS is rapidly and actively pumped inside the cell, clearing the intracellular space and protecting nNOS from removal by washing. Cell boundary is now visible because the cytoplasm is stained by released nNOS. 3D cross sections confirm that labeled nNOS is inside the cells. Yellow circles inside the cells are labeled endosomes.

Proteins tagged with a variety of cell penetrating peptides (CPPs) have been used to manipulate the interior of cells in culture and in situ for more than a decade (1-19). Our innovation is the use of coupling proteins that make strong protein-protein interactions to provide a convenient and powerful method to perturb cell interiors with a broad palette of selectively membrane permeable probes. Common and cheaply produced coupling proteins can be modified by introducing a CPP tag, enabling any protein that it binds to be moved into cells. It is relatively easy (and safe) to express and purify proteins with a tag that allows them to bind to a coupling protein with high affinity. As discussed later, some tags allow rapid purification of the protein chosen for delivery using a one-step affinity column.

Delivery of proteins to the interior of cells has many applications. In addition to mapping the location of the components of living cells with fluorescent tags, the availability of a system capable of translocating proteins into the cell interior can enable detection of internal components in real time in living cells, and provide tools for the manipulation of signaling pathways and gene expression by allowing the introduction of constitutively active kinases, repressors, and enhancers. Virus detection and destruction inside cells is a long term possibility, as are medical applications based on altering the metabolic state and/or expression profiles of cells.

Cell Penetrating Peptides.

Over the last decade a number of peptides have been discovered or designed that are rapidly internalized by mammalian cells. Cell-penetrating peptides (CPPs) are capable of mediating penetration of the plasma membrane, allowing the delivery of macromolecular cargoes to the interior of cells (1, 2, 3). CPPs are typically 10 to 30 amino acids long. The three major categories are arginine-rich, amphipathic and lysine-rich, and hydrophobic (4). CPPs have been attached to the N and C termini of payload proteins, and to intermediate positions using a variety of chemical conjugation strategies (e.g., targeting cysteine thiols).

While the uptake of CPPs by cells is well established, the mechanism is somewhat controversial, and several pathways appear to be in use (5). In part, this reflects differences among the peptides, but there are indications that the same peptide may be taken up by different pathways under different circumstances. The initial interaction of CPP-protein constructs with cellular membranes is through interactions with hydrophobic components and/or negatively charged groups (phospholipids, heparin sulfate proteoglycans) on the membrane surface (see FIG. 1). Uptake of CPP bound payloads proceeds via binding to membrane and invagination. FIG. 1-A shows CPP tagged payload is in medium outside cell membrane. FIG. 1-B shows CPP tagged payload binds to specific sites on the cell membrane. FIG. 1-C shows binding induced invagination in membrane. Depending on CPP tag, payloads can be targeted to internal compartments (nuclei, mitochondria) or cytoplasm (FIG. 1-D). The membrane associated, but not yet translocated, constructs are difficult to distinguish from translocated groups except by advanced 3D methods (e.g., confocal microscopy), which has led to artifacts in the study of CPP mechanisms. Once associated with the membrane surface, it is possible for several translocation mechanisms to come into play (6). Evidence for clathrin dependent endocytosis, caveolin dependent endocytosis, and macropinocytosis has been presented for different combinations of CPP and cargo molecule, e.g., protein, nucleic acid, drug (5-8).

Since the initial discovery of the Tat peptide (TaTp) in 1988 (6), a variety of CPPs have been found to enable the transport of macromolecular cargoes to cells in culture and within living animals (1, 2, 3). A number of well characterized CPPs originated from the N or C termini of viral proteins; these include TATp, oligoarginines (6, 8), MPG peptides, Pep1 (9, 10) and VP22(11). The TAT CPP derived from the carboxy terminus of the dopamine transporter is capable of enabling the translocation of large cargoes, and synthetic CPPs such as Xentry (12) (a short (LCLRPVG) peptide based on the N terminal region of Hepatitis B X protein) are capable of carrying very large proteins across cell membranes.

An example is the 1,024 amino acid of E. coli 3-galactosidase, which exists as a 464-kDa homotetramer. Each unit of 3-galactosidase subunit is a modular protein of five domains. These include a jelly-roll type barrel, two fibronectin type III-type barrels, a [3-sandwich domain, and a TIM-type barrel domain that contains the catalytic site. The ability of the CPP tag to enable translocation of an enormous multimer of modular components indicates that versatile translocation systems can be designed that use CPP tags to produce novel systems to manipulate the interior of cells.

Numerous patents have been granted for uses of cell penetrating peptides, but the use of a stable adaptor to couple the peptide to cargo has potential advantages in safety, utility, and in ease of purification of cargo. The use of the word ‘adaptor’ in the literature and in a few prior patent applications refers to the CPP itself, not to a coupling intermediate as described here.

Representative CPPs
Peptides	Origin	Sequences	Cargo types
Lysine rich CPPs and others derived from translocation domains
Tat	HIV-Tat protein	PGRKKRRQRRPPQ	Protein/peptide/siRNA/
			liposome/nanoparticle
Pen etratin	Homeodomain	RQIKIWFQNRRMKWKK	peptide/siRNA/liposome
Transportan	Galanin-	GWTLNSAGYLLGKINLKALAALAKKIL	Protein/peptide/siRNA
	mastoparan
Dat	Dopamine	FREKLAYIAP	Protein/peptide/siRNA
	transporter
VP-22	HSV-1	DAATATRGRSAASRPTERPRAPAR-	Protein
	structural	SASRPRRPVD
Amphipathic peptides
MPG	HIV Gp41-SV40	GALFLGFLGAAGSTMGAWSQPKKKRKV	siRNA/ODN/plasmid
Pep-1	Trp-rich motif-	KETWWETWWTEWSQPKKKRKV	Protein/peptide
	SV40 NLS
MAP	Chimeric	KALAKALAKALA	Small molecule/plasmid
SAP	Proline-rich	VRLPPPVRLPPPVRLPPP	protein/peptide
	motif
PPTG1	Chimeric	GLFRALLRLLRSLWRLLLRA	Plasmid
Arginine rich and other cell-penetrating peptides
Oligoarginine	Chimeric	Agr8 or Arg9	Protein/peptide/siRNA/ODN
hCT (9-32)	Human	LGTYTQDFNKTFPQTAIGVGAP	Protein/plasmid DNA
	calcitonin
SynB	Protegrin	RGGRLSYSRRRFSTSTGR	Doxorubicin
Pvec	Murine VE-	LLIILRRRIRKQAHAHSK	Protein/peptide
	cadherin
CPP, cell-penetrating peptide; NLS, nuclear localization sequence; PNA, peptide-nucleic acid; Tat, transcription-transactivating. See refs (1-19)

CPP Tagged Adaptor Proteins.

The inventor here discloses the production of CPP tagged adaptor proteins capable of interacting with a wide variety of payloads. Adaptors are ideally small, stable and easily purified proteins capable of interacting strongly with the payload, either via intrinsic protein-protein interactions or via a ligand (e.g., a covalently attached group such as biotin). This strategy has several advantages. It provides a unified strategy that allows a payload protein to be purified by affinity chromatography using an N or C terminal extension, and the same extension can be used to mediated binding to the CPP tagged adaptor/carrier.

The strategy allows the production of payloads with only a single tag, rather than a CPP tag and an affinity tag. It also means that only a few CPP tagged adaptors need to be developed to deliver many different payloads. This is significant because the CPP tagged versions of many potential payloads carry a potential risk to workers involved in their purification due to the cell membrane permeability enhancement. Production of a limited number of relatively benign adaptor proteins under well-controlled conditions provides a significant safety factor, and the adaptor-payload complex need only be assembled at the point of use, in cases where complex formation is much faster than uptake by cells even being added separately to cell cultures.

The adaptor-payload complex can be designed to dissociate on internalization (see FIG. 2). One convenient way of doing this is to use an adaptor that responds to the internal cellular conditions, although other methods (an unstable linkage, autocatalytic dissociation, photodissociation) are also possible. The use of calcium by mammalian cells as a signal provides a promising avenue for release of payloads; cell interiors are normally maintained at very low levels of calcium by ATP driven pumps, and cells contain a variety of calcium biosensors that respond to transient increases in calcium to tightly bind and release target peptides. In a preferred embodiment, the adaptor protein is a calcium biosensor such as calmodulin. FIG. 2-A shows the association mediated by protein-protein or protein-ligand interactions of CPP tagged adaptor and cargo with adaptor ligand into binary complex. FIG. 2-B shows binding to the cell membrane. FIG. 2-C shows internalization. FIG. 2-D shows redistribution to internal compartments.

CPP Calmodulin.

Calmodulin is a multifunctional calcium biosensor that folds into a dumbbell-shaped configuration in the presence of calcium (20,21). The ends of the dumbbell each contain two calcium binding EF hands. The alpha helix that connected the two globular regions breaks and closes around targets containing a 17 amino acid canonical motif or one of several alternative target motifs. Binding of CaM to targets is high affinity (picomolar) and is typically diffusion limited. CaM is a major mediator of calcium signaling in mammalian cells, and is the archetypical member of the EF hand—calmodulin superfamily of calcium signaling proteins. Calmodulin is small (16.7 kDa), soluble, and remarkably heat resistant. It is easy to produce site directed mutants and chimeras with calmodulin. The production of novel calmodulin constructs has the potential to provide unique and valuable reagents for cell biology research.

TAT peptides are short signal sequences that mediate transport of proteins across the membranes of many cells. Although TAT peptides were initially believed to work by directly mediating transport across phospholipid bilayers, they can drive the uptake of large proteins that could not cross the membrane without an active uptake process. It now appears that TAT peptides attach to receptors on the membrane and cause internalization in coated pits (5,15,17). Several patents have been granted for constructs that can be internalized by processes that rely on recognition of short TAT peptides attached as C or N terminal fusions.

Since the peptides are covalently attached through the peptide backbone, cargo remains attached to the CPP in cell interior. In addition, cargo proteins must be purified as CPP adducts. This means that expression in eukaryotes is complicated by binding to import machinery via the CPPs, and handling of the material is complicated because many desirable products are rendered potentially hazardous by the CPP tag.

The invention greatly extends the usefulness of TAT peptide constructs (and related CPP constructs) by expressing TAT fusions of small proteins that strongly bind other proteins. The inventor has designed a TAT calmodulin which is readily taken up by cells in culture (initially CHO cells) and should be taken up by cells in whole organisms. TAT was used as the initial CPP tag as the initial tag because of prior success in producing TAT tagged proteins that are taken up by mammalian cells, but other CPP tagged calmodulins are in production.

Initially, TAT tagged calmodulin was produced exactly as purify His-tagged calmodulin using His tag and nickel column. TAT tagged calcium biosensors can be purified using a column decorated with peptides recognized by the biosensor. For calmodulin, this is a 17 amino acid canonical sequence bound with high affinity in the presence of calcium. This will allow us to make calmodulin without the His tag by affinity chromatography, binding to the column in the presence of calcium and eluting with the calcium ionophore EDTA.

In a preferred embodiment, the payload delivered by the CPP tagged adaptor is a modulator (activator or repressor) of transcription. In another preferred embodiment, the payload is a probe that measures a property of the cell interior (e.g., an oxidation monitor, NO sensors, pH sensor). In another preferred embodiment, the payload is a kinase, phosphatase or other enzyme, which may be modified to be constitutively active.

Other payloads, including liposomes and their contents, nucleic acids, inhibitors, and drugs can also be delivered by extensions of the same methods (e.g. using DNA binding proteins with calmodulin binding N or C terminal extensions. In a preferred embodiment, the payload is a nucleic acid delivered using a DNA or RNA binding protein with an adaptor recognition tag. In another a preferred embodiment, the payload is a drug or other small molecule delivered using a protein or other scaffold that binds the small molecule and is equipped with an adaptor recognition tag.

Tagged GFP and Other Fluorescent Probes.

Green Fluorescent Protein (GFP) and its engineered variants are powerful tools for the labeling of cell interiors. GFP is typically expressed after transfection with the appropriate vector, but many cell types are resistant to transfection. In a preferred embodiment, the payload delivered is a fluorescent probe such as a GFP fusion containing a site that recognizes an internal target and a tag recognized by a CPP adaptor (e.g., a calmodulin binding peptide recognized by TAT-CaM). GFP can be relatively easily purified, useful fluorescent probes are not limited to GFP and its homologs. They are widely used in part because they can be expressed in mammalian cells after transfection with a shuttle vector, and spontaneously generate a fluorophore inside the cells. The ability to deliver external probes broadens the possibilities.

A wide variety of proteins can be labeled with commercially available custom fluorophores (e.g., the extensive series sold by Alexa) and introduce them into the interior compartments of cells with CPP tags. This allows investigators to follow the tagged proteins in the cell with confocal microscopy, but also to conduct more demanding experiments, including FRET (fluorescence resonance energy transfer) and fluorescence lifetime experiments (see FIG. 3). As shown in FIG. 3, excitation of D leads to emission from A only when A and D are in proximity due to complex formation. This also reduces emission from the donor. Examples of donor acceptor pairs with good overlap include Alexa Fluor 488 and Alexa Fluor 647.

In FRET experiments, components are labeled with fluorophores chosen so that the emission spectrum of one (the donor) is heavily overlapped with the excitation spectrum of the other (the acceptor). If the labeled molecules associate in the cell, Forster energy transfer will cause the acceptor to fluoresce when the donor is excited by pumping its absorbance lines. This provides information about complex formation in cells.

In lifetime experiments, a fluorophore is repeatedly excited by a pulse from a laser and the fluorescence decays are collected, yielding the lifetimes of the fluorophore in all environments. Typically three or four environments can be readily distinguished with lifetimes in the 50 ps to 5 ns range and contributions as low as a few percent.

FRET experiments can be carried out inside cells using two different GFP variants, but using CPP adaptors to deliver a pair of proteins labeled with different synthetic fluorophores would be advantageous for several reasons. Paired fluorophores optimized for FRET are sold by Alexa and DyLight. These have far better properties (e.g., yield and spectral overlap) than the engineered GFP variants. An important advantage is that they are small and introduce much less steric interference than a GFP fusion.

Calmodulin and the EF Hand Proteins.

Calmodulin is remarkable for its high sequence conservation; only four other proteins are more conserved in eukaryotes. Mammalian calmodulins are identical, and the C. elegans protein is 96% identical to its human homolog. The sequence homology of calmodulin is not imposed primarily by the requirement for calcium binding and the associated organization into the characteristic dumbbell shape (FIG. 4); this could be accomplished by far lower levels of similarity. Instead, the primary driver of conservation is the retention of target specificity. Since calmodulin binds to many Ca²⁺ activated targets in cells, the ability of the targets and calmodulin to co-evolve is severely restricted. FIG. 4-A shows the structure of Ca²⁺-CaM bound to a canonical target peptide in the center of the molecule and FIG. 4-B shows the structure in the dumbbell-shaped conformation in the absence of target. The central helix breaks during recognition and binding, allowing calmodulin to wrap around the target. Ca²⁺ are shown as small speres; the protein is less ordered in the absence of Ca²⁺ (not shown). Free N and C termini are visible.

As shown in the alignment below, sequence similarity within the calmodulin-EF hand superfamily is much lower; identity within the four human sequences shown is 20%. The sequence variation within the superfamily allows the members to recognize and regulate distinct targets in response to a single ionic signal. It allows us to make use of the different specificity of superfamily members to produce EF hand based adaptors that are specific to different target sequences (22, 23); all these targets are roughly 17 AA in length because of the dimensions of the folded EF hand proteins, but the amino acid sequences of the targets are different. (There are different binding modes for some targets, but this is not important for our purposes). This is important in the long run because it confers potential to address different payloads to different cellular compartments (10).

Structures of calcium-calmodulin bound to a canonical target peptide (left) and in the dumbbell-shaped conformation in the absence of target (right). The central helix breaks during recognition and binding, allowing calmodulin to wrap around the target. The protein is less ordered in the absence of calcium (not shown).

Delivery of Payloads with CPP Tagged Calmodulin.

Good evidence has been obtained for delivery of target proteins to the interior of cells with CPP labeled calmodulin. The initial demonstrations were designed to use neuronal nitric oxide synthase (24) and CaM Kinase (25); both enzymes are activated by calcium/calmodulin, and both can be purified on a calmodulin column. CaM kinase isoforms have monomer molecular masses of ˜41 kDa; the truncated CaM kinase II sold by New England Biolabs has a molecular mass of 36 kDa. However, CaM kinases form very large quartenary complexes of 400-600 kDa, making them an exacting test for the calmodulin mediated translocation system, comparable to beta-galactosidase. The nNOS active dimer has a molecular mass of ˜322 kDa. Both proteins can be readily labeled with high quantum yield fluorophores that have distinctive spectral signatures, allowing their uptake and cellular distribution to be readily evaluated.

These proteins were chosen because they contain a calmodulin binding motif, but most proteins can be produced with a small calmodulin binding tag at the N or C terminus without significantly affecting their activity, or like neuronal nitric oxide synthase (nNOS) with an internal tag associated with an exposed surface loop.

An obvious alternative is the attachment of a CPP directly to the payload. Numerous patents cover the use of various CPPs attached to payloads by covalent or in a few cases non-specific non-covalent interactions. There are several drawbacks: this requires additional handing of potentially toxic CPPs, and the CPP would remain on the tag after internalization.

In one embodiment of the current invention, payloads are tagged with an adaptor recognized moiety (e.g., a calmodulin binding peptide) using standard cross linking methods (see FIG. 7). FIG. 7-A shows nNOS added without CPP adaptor. Background still shows stained nNOS after washing with media. Some nNOS adheres to the cell surface; 3D cross sections show no nNOS inside cells. FIG. 7-B shows nNOS in the presence of TAT-CaM. A huge amount of nNOS is rapidly and actively pumped inside the cell, clearing the intracellular space and protecting nNOS from removal by washing. Cell boundary is now visible because the cytoplasm is stained by released nNOS. 3D cross sections confirm that labeled nNOS is inside the cells. Yellow circles inside the cells are labeled endosomes. For proteins that are produced by investigators themselves, it removes the advantages of integrated affinity purification and CPP-adaptor attachment. Nevertheless, for some applications the direct coupling approach could prove to be a useful alternative. For example, commercially obtained proteins with no CaM binding site can readily be tagged and rendered cell permeable this way.

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Claims

1. A biological complex for translocating a cargo into a cell, the complex comprising: a. a cell penetrating peptide (CPP) linked to an adaptor by a covalent linkage, andb. a cargo comprising an adaptor binding molecule, wherein the adaptor binding molecule reversibly binds to the adaptor such that the cargo is released from the adapter upon exposure to an intracellular environment;wherein the adaptor is calmodulin and the adaptor binding molecule is a calmodulin binding peptide.

2. The complex of claim 1, wherein the CPP is selected from the group consisting of TAT, Penetratin, Transportan, Dat, VP-22, MPG, Pep-1, MAP, SAP, PPTG1, oligoarginine, hCT (9-32), SynB, and Pvec.

3. The complex of claim 1, comprising SEQ ID NO: 22 and SEQ ID NO: 1.

4. The complex of claim 1 where the cargo is a fluorescence tagged Fab antibody fragment.

5. A method for delivering a cargo into a cell, the method comprising contacting the cell with a biological complex under conditions suitable for translocating the biological complex into the cell, the biological complex comprising: a. a cell penetrating peptide (CPP) linked to an adaptor by a covalent linkage, andb. a cargo comprising an adaptor binding molecule, wherein the adaptor binding molecule reversibly binds to the adaptor such that the cargo is released from the adapter upon exposure to an intracellular environment;wherein the adaptor is calmodulin and the adaptor binding molecule is a calmodulin binding peptide.

6. The method of claim 5, wherein the CPP is selected from the group consisting of TAT, Penetratin, Transportan, Dat, VP-22, MPG, Pep-1, MAP, SAP, PPTG1, oligoarginine, hCT (9-32), SynB, and Pvec.

7. The method of claim 5, wherein the cargo is used for internal measurements of cell conditions.

8. The method of claim 5, wherein the cell conditions are selected from the group consisting of pH, calcium, oxygen, nitric oxide concentration, and combinations thereof.

9. The method of claim 5, wherein the cargo is delivered to detect or measure the presence of intracellular targets.

10. The method of claim 5, wherein the cargo comprises an antibody or a portion thereof.

11. The method of claim 5, wherein the cargo performs a function selected from the group consisting of perturbation of a cellular state, modification of cellular expression, modification of a cellular genome of an organism, treatment of a disease, and combinations thereof.

12. The method of claim 5, wherein the calmodulin binding peptide is an N or C terminal extension.

13. The method of claim 5, wherein the adaptor contains a phosphorylation site, and upon exposure to endogenous kinase and/or phosphatase activity of the cell, the cargo is released.

14. The method of claim 5, wherein the cell is in vitro.

15. The method of claim 5, wherein the cell is in vivo.

16. The method of claim 5, wherein the cargo is purified by affinity column chromatography.

17. The method of claim 5, wherein the cargo is delivered to an internal cellular compartment.

18. The method of claim 5 performed in vivo or in situ in cultured cells or in isolated cells.

19. The method of claim 5, wherein the cargo is a fluorescence tagged Fab antibody fragment.