Delivery agent

Abstract

A composition comprising a polycationic agent and a polyanionic agent, and kits comprising the composition, is provided. In embodiments, the polyanionic agent is a nucleic acid and the polycationic agent is a modified polyalkyleneimine polymer.

Claims

1. A composition for delivering a peptide or protein into a cell, which composition comprises a polycationic agent and a polyanionic agent, wherein the polyanionic agent is a nucleic acid and the polycationic agent is a modified polyalkyleneimine polymer having a repeat unit of formula I or formula V: ##STR00014## where: Z1 and Z2 each independently represent H, alkyl or a branching chain; Y1 and Y2 each independently represent a group having formula II or III: ##STR00015## where: X1 is selected from the group consisting of H, -alkyl, SH, CN, NH.sub.2, N(alkyl).sub.2,NH(alkyl), CONH.sub.2-, ##STR00016## NHCONH.sub.2, X2 is selected from the group consisting of CH.sub.2, O, S, NH, or N(alkyl)-; provided that at least one of X1 and X2 is a hydrophilic group; m1, m2, n1 and n2 each independently is 0, 1, 2or 3; and p1 and p2 each independently is 1 or 2.

2. The composition of claim 1, wherein the nucleic acid is DNA.

3. A kit for delivering a peptide or protein into a cell, which kit comprises a polycationic agent in a first container and a polyanionic agent in a second container, wherein the polyanionic agent is a nucleic acid and the polycationic agent is a modified polyalkyleneimine polymer having a repeat unit of formula I or formula V: ##STR00017## where: Z1and Z2 each independently represent H, alkyl or a branching chain; Y1 and Y2 each independently represent a group having formula II or III: ##STR00018## where: X1 is selected from the group consisting of H, -alkyl, SH, CN, NH.sub.2, N(alkyl).sub.2,NH(alkyl), CONH.sub.2-, ##STR00019## CONH.sub.2, NHCONH.sub.2, X2is selected from the group consisting of CH.sub.2, O, S, NH, or N(alkyl)-; provided that at least one of X1 and X2 is a hydrophilic group; m1, m2, n1 and n2 each independently is 0, 1, 2or 3; and p1 and p2 each independently is 1or 2.

4. The kit of claim 3 where the nucleic acid is DNA.

5. A method for delivering a peptide or protein into a target cell, which method comprises contacting the peptide or protein with a polycationic agent and a polyanionic agent to form a complex and contacting the complex with the target cell so as to deliver the peptide or protein thereto, wherein: (1) the polyanionic agent is a nucleic acid; and (2) the polycationic agent is a modified polyalkyleneimine polymer having a repeat unit of formula I or formula V: ##STR00020## where: Z1 and Z2 each independently represent H, alkyl or a branching chain; Y1 and Y2 each independently represent a group having formula II or III: ##STR00021## where: X1 is selected from the group consisting of H, -alkyl, SH, CN, NH.sub.2, N(alkyl).sub.2, NH(alkyl), CONH.sub.2, ##STR00022## NHCONH.sub.2, X2is selected from the group consisting of CH.sub.2, O, S, NH, or N(alkyl)-; provided that at least one of X1 and X2 is a hydrophilic group; m1, m2, n1and n2 each independently is 0, 1, 2 or 3; and p1 and p2 each independently is 1or 2.

6. The method according to claim 5, wherein the peptide or protein is contacted with the polyanionic agent prior to contacting the polycationic agent.

7. The method according to claim 5, wherein the peptide or protein comprises an enzyme, an antibody or an inert protein.

8. The method according to claim 5, wherein the target cell is a suspension cell, an adherent cell, a primary cell or cultured cell.

9. The method of claim 5 where the nucleic acid is DNA.

Description

(1) The invention will now be described in further detail, by way of example only, with reference to the accompanying drawings in which:

(2) FIG. 1 shows a comparison between embodiments of the invention and the prior art as requested by transfection efficiency and mean fluorescence intensity when delivering labelled antibody to HeLa cells;

(3) FIG. 2 shows a comparison between embodiments of the invention and the prior art as measured by distribution of transduced -galactosidase in cytoplasmic and membrane protein fractions;

(4) FIG. 3 shows a comparison between different polyanions in relation to the enhancement of cationic polymer mediated protein transfection as measured by transfection efficiency and mean fluorescence intensity;

(5) FIG. 4 shows a comparison between different polyanions in the enhancement of protein transfection efficiency by polycations pHP and LPEI, as measured by transfection efficiency and mean fluorescence intensity;

(6) FIG. 5a shows polyanion enhancement of pHP-mediated peptide and inert protein transfection as measured by transfection efficiency and mean fluorescence intensity;

(7) FIG. 5b shows the enhancement of transfection by polyanion PWp of proteins having different pI as measured by transfection efficiency and mean fluorescence intensity;

(8) FIG. 6 shows polyanion enhancement of pHP-mediated protein transfection into primary and suspension cells as measured by transfection efficiency, toxicity and mean fluorescence intensity; and

(9) FIG. 7 shows the effect of component mixing on protein transfection as measured by transfection efficiency, toxicity and mean fluorescence intensity.

DETAILED DESCRIPTION OF THE INVENTION

(10) Transfection efficiency of Polyhydroxypropyleneimine (pHP) was initially tested on HeLa cells using Alexa Fluor (AF) 488-labeled antibody (goat IgG) as a control protein. Different polyanions: DNA, sodium polyphosphate (pP) and sodium polytungstate (pW) were tested as additives aiming to improve complex formation. Commercial protein transfection reagents Chariot (Ambion) and ProJect (Pierce) were used as positive controls. The transfection efficiency was evaluated using three criteria: the percent of AF488 positive cells, the percent of dead cells (toxicity) and the mean fluorescence intensity (MFI).

(11) The results show that antibody cannot internalize into the cell on its own (FIG. 1). The amount of AF488-positive cells increased to 65% when cationic polymer pHP was used in complex with the antibody. The percent of transfected cells was even higher (up to 85-95%) when different polyanions (DNA, pP or pW) were added into the mixture, suggesting that polyanions have positive effect for protein delivery. Polyanions alone, on the other hand, have no effect on protein entry into the cell (FIG. 3). Comparison of obtained antibody transfection results with two commonly used commercial protein transfection reagentsChariot and Project, reveals very similar transfection efficiencies (85% for Chariot and 80% for Project). However, when comparing the MFI values, the polytungstate evidently is more effective and mediates the biggest amount of protein (MFI 370) being delivered into the cell, which is significantly higher than that shown for Chariot or ProJect (MFI100 and 150, respectively).

(12) To determine cellular localization of transduced proteins, the cells were transfected with -galactosidase using the same compositions and protocols as described above. The cells were further fractionated using ProteoJET Membrane Protein Extraction Kit (Fermentas) in order to separate membrane and cytosolic proteins. Enzymatic activity of -galactosidase was estimated in both fractions (FIG. 2). Results show that majority of -gal activity was detected in the cytosolic fraction for all pHP and Project-mediated transfections, while very little or no -gal activity was detected in the membrane fraction of pHP-transfected cells, suggesting that cationic polymer (with or without polyanions) positions transduced proteins exclusively inside the cell. For Project, however, considerable amount of -gal activity was found in the membrane, indicating that equivalent amount of protein after transfection remains stuck within or on the surface of the cellular membrane. For Chariot-mediated transfection, significantly more -galactosidase was found in the membrane fraction than in the cytosol. In conclusion, pHP-polyanion mixture facilitates highly efficient protein transduction resulting primarily in cytosolic protein localization inside the cell.

(13) To examine if other polyanions contribute to cationic polymer-mediated protein transduction, we tested sodium phosphomolybdate hydrate (pMoP), ammonium molybdate tetrahydrate (pMo), as well as sodium phosphotungstate tribasic hydrate (pWP) along with previously used polyanions: DNA, pP and pW (FIG. 3). The results show that all analyzed polyanions enhance protein transfection to a similar level of 80-95%. The MFI data, however, singled out polytungstates (with or without hetero atoms) as the most potent enhancers (MFI1200). The polyoxometalates (POMs) carrying hetero atom (pWP and pMoP) apparently performed slightly worse than POMs without hetero atom (pW and pMo).

(14) To further investigate if polyanions have positive effect in combination with other polycations used in protein transfections, a popular cationic polymerLPEI was tested along with pHP in fluorescently labelled antibody transfections. PolyanionsDNA, pP and pW were used to assist protein packaging prior to complexation with LPEI (FIG. 4). The results show that polyanions enhance LPEI-mediated protein transfection as efficiently as pHP-mediated transfection. The MFI values increase from 10 units (protein-LPEI) to 25, 60 and 130 units upon addition of DNA, pP or pW, respectively. The results suggest that negatively charged polyanions may interact with positively charged regions of the antibody and consequently facilitate protein-polyanion interaction with the positively charged polycation.

(15) To demonstrate that polyanions are able to enhance transfection of any type protein, a number of proteins of different size, pI value or function were chemically conjugated to FITC and examined using the same conditions as those used for antibody transfections described above. Successful delivery of 5 kDa peptide (FIG. 5a), 12 kDa cytochrome C, 18 kDa -lactoglobulin (FIG. 5b), 66 kDa BSA, 97 kDa amyloglucosidase, as well as earlier tested 116 kDa -galactosidase (FIG. 2) and 150 kDa IgG (FIG. 1, 3, 4), confirm that polyanions enhance transfection of any size protein carried by cationic polymer pHP. The MFI values primarily depend on the size of the protein, i.e. the extent of FITC labelling. Smaller proteins had lower number of FITC molecules and, as a result, their fluorescence was weaker. The transfection of proteins bearing different pI (amyloglucosidasepI 3.5, -lactoglobulin pI 5.5, cytochrome C pI 10.5) gave similar results (FIG. 5b), all three proteins were delivered with 90% efficiency. The amount of polyanion used in this case, depended on the pI of the protein: less polyanion was needed for transfection of amyloglucosidase (0.5 l), more for transfection of cytochrome C (1.0 l). Overall, the results show, that polyanion-polycation combination enhances the transfection of proteins with (i) different size, (ii) different pI and (iii) different function.

(16) For the final evaluation of polyanion exerted effect on the pHP-mediated protein transfection the experiments were carried on different cell types: primary human lung fibroblasts (primary cellsusually difficult to transfect), HeLa S3 (loosely adherent cell line), and Jurkat T cell lymphoma cells (suspension cell lineknown to be very difficult to transfect by chemical methods). The results showed that irrespective of the cell type used, the transfection efficiencies reached 90% (FIG. 6). The fluorescence level in strongly adherent HLF cells was the highest (MFI 1500), while in semiadherent or suspension cells, HeLa S3 and Jurkat, the MFI was 350 and 500, respectively, suggesting that the extent of macromolecule uptake depends on the cell type. Cell size in this experiment should be taken into consideration as well, since HLF cells are significantly bigger, can internalize more material than HeLa S3 or Jurkat cells, and thus fluoresce more intensively than smaller cells. In conclusion, the polyanion (here, pWP) grouping with protein prior to complexation with polycation (pHP) facilitates efficient protein delivery into the primary, adherent and suspension cell types.

(17) Evaluation of polyanion-protein-polycation complex formation after different component mixing schedule and its influence on transfection efficiency was carried out in order to determine the best possible way to form protein-pHP complexes and to ensure the most efficient cargo transport through the cellular membrane. The results apparently were very similar, no matter how the components were mixed together (FIG. 7), the transfection efficiencies ranged from 80 to 95%. Slightly lower MFI values (200 units) suggested that protein should not be the last element added into the mix, but rather mixed with either polycation or polyanion first.

Example 1

Analysis of the Protein Transfection Using pHP

(18) Transfection of HeLa (Human cervical carcinoma-derived cell line) cells was carried out as follows: one day before the transfection experiment, the cells were seeded in a 24-well tissue culture plate at the density of 510.sup.4 cells per well in the total volume of 1 ml DMEM culture medium supplemented with 10% FBS. The cells were incubated at 37 C. in a CO.sub.2 incubator until they reached 70-80% confluency (usually within 24 h). On the day of transfection, the growth medium was removed and replaced with 0.5 ml of warm serum-free medium. Alexa Fluor 488-labeled goat IgG antibody (1 g) was diluted in 100 l of 0.15M NaCl solution and mixed with different amounts of polyanions: DNA (1 g pUC18), sodium polyphosphate (10 mM pP1 l) or sodium polytungstate (10 Mm pW3 l). Cationic polymer pHP (1 l) was added into the protein-polyanion mixture and vortexed immediately for few seconds to ensure even distribution of the material. The complexes were allowed to form for 15-20 min at room temperature and added to the cell culture in a drop-wise manner. The cells were further incubated for 2 h at 37 C. in a CO.sub.2 incubator. To remove unincorporated complexes, the cultures were rinsed with PBS, and the cells were analyzed by FACS (Fluorescence Activated Cell Sorter). Transfections using Chariot (Ambion) and ProJect (Pierce) reagents were carried out following manufacturer suggested protocols.

Example 2

Analysis of the Protein Localization after Polyanion-Polycation Mediated Transfection

(19) HeLa cell transfection with -galactosidase (1 g) was carried out using the same protocol and conditions as described above. The cells were further fractionated using ProteoJET Membrane Protein Extraction Kit (Fermentas) in order to separate membrane and cytoplasmic proteins. The enzymatic activity of -galactosidase in both fractions was estimated using colorimetric assay.

Example 3

Analysis of Different Polyanions in pHP-Mediated Protein Transfections

(20) Several different polyanions were tested for their ability to improve labelled-IgG transfection. Polyanions were grouped as follows: (i) phosphatesheterophosphates (DNA) and homophosphates (sodium polyphosphatepP), (ii) POMswithout hetero atom (sodium polytungstate, pW, or ammonium molybdate tetrahydrate, pMo) and with hetero atom (sodium phosphotungstate tribasic hydrate, pWP, and sodium phosphomolybdate hydrate, pMoP). HeLa cells were prepared for transfection essentially as described in Example 1. The amount of each polyanion used was: 3 l of pW, pWP or pMoP, and 2 l of pMo (each 10 mM stock concentration), 0.5 l of pP (30 mM stock concentration), 1 g of DNA. Polyanion-Antibody-pHP mixtures were incubated for 15 min and added to the cells in a drop-wise manner. Transfection results were processed 2 h later using Guava Easy Cyte Plus flow cytometry system (Millipore).

Example 4

Analysis of Polyanions in Different Polycation-Mediated Protein Transfection

(21) Cationic polymer LPEI (ExGen 500) was tested in Alexa Fluor 488-labeled goat IgG transfection using polyanionsDNA, pP and pW to assist the protein packaging prior to complexation with LPEI. Chinese hamster ovary cells (CHOk1) were prepared for transfection essentially the same way as HeLa cells (example 1). The cells were cultured in RPMI medium supplemented with 10% FBS, the transfection was carried out in serum free RPMI medium. The complexes were formed the same way as described in example 1 for pHP, the amount of LPEI used3.3 l.

Example 5

Analysis of Polyanions in Transfections of Proteins of Different Size and pI

(22) FITC-labeled proteins5 kDa peptide, BSA, amyloglucosidase (pI 3.5), -lactoglobulin (pI 5.5) and cytochrome-C (pI 10.5) were transfected into HeLa cells following the procedure described in Example 1. The amount of pWP used: 0.5 l for amyloglucosidase and -lactoglobulin, 1 l for cytochrome C.

Example 6

Analysis of the Polyanion Effect on Difficult to Transfect Cell Lines

(23) Comparison of protein transfer efficiency using pHP and polyanions (pWP) was tested in suspension cell lines, HeLa S3 and Jurkat (Human T cell lymphoma cell line), as well as in primary cells HLF (human lung fibroblasts). Suspension cells were seeded at the density of 210.sup.5 cells/well, HLF 510.sup.4/well 24 hours before the transfection. Antibody-pHP complexes in 0.15 M NaCl solution were prepared as described earlier.

Example 7

Analysis of the Component Mixing Order Effect on Protein Transfection

(24) HeLa cells were prepared for transfection as described in Example 1. The antibody IgG (1 g)pWP (1 l)pHP (1 l) complexes were prepared in 0.15 M NaCl following different component mixing order: IgG+PA+pHP, PA+IgG+pHP, IgG+pHP+PA, pHP+IgG+PA PA+pHP+IgG, and pHP+PA+IgG. Complexes were allowed to form for 15 min and added to the cells in a drop-wise manner.