Amphipathic peptide
09744244 · 2017-08-29
Assignee
Inventors
- Helen McCarthy (Belfast Antrim, GB)
- Aleksey Zholobenko (Larchwood Staffordshire, GB)
- Ashley Davison (Belfast Antrim, GB)
- Tracy Robson (Belfast Antrim, GB)
Cpc classification
A61K47/6455
HUMAN NECESSITIES
C12Y114/13039
CHEMISTRY; METALLURGY
A61K48/0025
HUMAN NECESSITIES
C12N15/87
CHEMISTRY; METALLURGY
A61K47/64
HUMAN NECESSITIES
C07K2319/33
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
C07K14/00
CHEMISTRY; METALLURGY
C12N2320/32
CHEMISTRY; METALLURGY
A61K31/713
HUMAN NECESSITIES
C07K2319/10
CHEMISTRY; METALLURGY
A61K47/60
HUMAN NECESSITIES
International classification
C07K14/00
CHEMISTRY; METALLURGY
A61K31/713
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The present invention is directed to an amphipathic peptide and methods of using the amphipathic peptide for delivering small molecule agents to a cell. Ideally, the amphipathic cell penetrating peptide comprises less than approximately 50 amino acid residues with at least 6 arginine residues, at least 12 Alanine Residues, at least 6 leucine residues, optionally at least one cysteine residue, and at least two but no greater than three glutamic acids wherein the arginine residues are evenly distributed along the length of the peptide; and the peptide has a defined ratio of arginine to negatively charged amino acid residues and a defined ratio of hydrophilic amino acid residues to hydrophobic amino acid residues. The present invention is also directed to a nanoparticle and cell delivery system comprising the amphipathic cell penetrating peptide of the invention. The peptide, nanoparticle or cell delivery system of the invention may be used in therapy. For example, the peptide may be used as a therapeutic agent delivery system, in which the therapeutic agent may include nucleic acids or other small molecules.
Claims
1. An amphipathic cell penetrating peptide of less than approximately 50 amino acid residues comprising one of the following amino acid sequences: TABLE-US-00009 (SEQ ID No. 1) WEARLARALARALARHLARALARALRACEA (SEQ ID No. 2) WEARLARALARALARLARALARALRACEA (SEQ ID No. 3) WEARLARALARALARLARALARALRACEA (SEQ ID No. 4) WEARLARALARALARELARALARALRACEA (SEQ ID No. 5) REARLARALARALARLARALARALRACEA (SEQ ID No. 6) REARLARALARALARLARALARALRAREA (SEQ ID No. 7) REARLARALARALARELARALARALRAREA or a fragment thereof, said fragment comprising at least 6 arginine residues (R), at least 12 Alanine Residues (A), at least 6 leucine residues (L), optionally at least one cysteine residue (C), and at least two but no greater than three glutamic acids (E) wherein the arginine (R) residues are evenly distributed at every third or fourth amino acid position along the entire length of the peptide; the ratio of arginine (R) to negatively charged glutamic acid (E) residues is from at least 6:2 to 9:2; and the ratio of hydrophilic amino acid residues to hydrophobic amino acid residues at pH 7 is at least 30:67 to 40:60.
2. A peptide according to claim 1 consisting of one of the following amino acid sequences TABLE-US-00010 (SEQ ID No. 1) WEARLARALARALARHLARALARALRACEA (SEQ ID No. 2) WEARLARALARALARLARALARALRACEA (SEQ ID No. 3) WEARLARALARALARLARALARALRACEA (SEQ ID No. 4) WEARLARALARALARELARALARALRACEA (SEQ ID No. 5) REARLARALARALARLARALARALRACEA (SEQ ID No. 6) REARLARALARALARLARALARALRAREA (SEQ ID No. 7) REARLARALARALARELARALARALRAREA.
3. The peptide according to claim 1, wherein said peptide is coupled to a polyethylene glycol (PEG) molecule.
4. The peptide according to claim 1 further comprising a cell targeting motif sequence conjugated to the peptide via a spacer sequence.
5. The peptide according to claim 1 comprising a cell targeting motif sequence conjugated to the peptide via a spacer sequence wherein the cell targeting motif is the metastatic prostate cancer targeting peptide TMTP-1 (NWRQ, SEQ ID No. 12) and the spacer is an alpha helical spacer comprising from 1 to 4 repeats of the sequence EAAAK (SEQ ID No. 13).
6. The peptide according to claim 1 comprising a cell targeting motif sequence conjugated to the peptide via a spacer sequence wherein the cell targeting motif is the metastatic prostate cancer targeting peptide TMTP-1 (NWRQ, SEQ ID No. 12) and the spacer is an alpha helical spacer.
7. A method for the nuclear localisation of nucleic acids, selected from DNA, RNA, shRNA, or siRNA, to cells comprising the steps of providing a peptide according to claim 1 complexed with a nucleic acid to form nanoparticles and administering to said cells.
8. A cell delivery system comprising an amphipathic cell penetrating peptide of less than approximately 50 amino acid residues complexed with a nucleic acid or other agent to form nanoparticles, wherein the other agent is a negatively charged or hydrophilic compound, said peptide comprising one of the following amino acid sequences: TABLE-US-00011 (SEQ ID No. 1) WEARLARALARALARHLARALARALRACEA (SEQ ID No. 2) WEARLARALARALARLARALARALRACEA (SEQ ID No. 3) WEARLARALARALARLARALARALRACEA (SEQ ID No. 4) WEARLARALARALARELARALARALRACEA (SEQ ID No. 5) REARLARALARALARLARALARALRACEA (SEQ ID No. 6) REARLARALARALARLARALARALRAREA (SEQ ID No. 7) REARLARALARALARELARALARALRAREA or a fragment thereof, said fragment comprising at least 6 arginine residues (R), at least 12 Alanine Residues (A), at least 6 leucine residues (L), optionally at least one cysteine residue (C), and at least two but no greater than three glutamic acids (E) wherein the arginine (R) residues are evenly distributed at every third or fourth amino acid position along the entire length of the peptide; the ratio of arginine (R) to negatively charged glutamic acid (E) residues is from at least 6:2 to 9:2; and the ratio of hydrophilic amino acid residues to hydrophobic amino acid residues at pH 7 is at least 30:67 to 40:60.
9. The cell delivery system according to claim 8 wherein the nucleic acid is one or more of DNA, RNA, shRNA, and siRNA.
10. The cell delivery system according to claim 8 wherein the nucleic acid is inducible nitric oxide synthase (iNOS) plasmid DNA under the control of a tumour specific promoter.
11. The cell delivery system according to claim 8 wherein the nucleic acid is inducible nitric oxide synthase (iNOS) plasmid DNA under the control of a tumour specific promoter selected from the human osteocalcin (hOC) promoter, osteopontin promoter, WAF1, CARG and a prostate specific promoter.
12. The cell delivery system according to claim 8 wherein the other agent is a small molecule agent.
13. The cell delivery system according to claim 8 wherein said small molecule agent is a phosphate based drug, selected from alendronate, etidronate, zolendrate or any other nitrogen or non-nitrogen based bisphosphonate drug.
14. The cell delivery system according to claim 8 wherein the other agent is gold.
15. The cell delivery system according to claim 8, said peptide consisting of one of the following amino acid sequences TABLE-US-00012 (SEQ ID No. 1) WEARLARALARALARHLARALARALRACEA (SEQ ID No. 2) WEARLARALARALARLARALARALRACEA (SEQ ID No. 3) WEARLARALARALARLARALARALRACEA (SEQ ID No. 4) WEARLARALARALARELARALARALRACEA (SEQ ID No. 5) REARLARALARALARLARALARALRACEA (SEQ ID No. 6) REARLARALARALARLARALARALRAREA (SEQ ID No. 7) REARLARALARALARELARALARALRAREA.
16. The cell delivery system according to claim 8 wherein the peptide comprises or consists of the following amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID No. 1).
17. The cell delivery system according to claim 8, wherein said peptide is coupled to a polyethylene glycol (PEG) molecule.
18. The cell delivery system according to claim 8, said peptide further comprising a cell targeting motif sequence conjugated to the peptide via a spacer sequence.
19. The cell delivery system according to claim 8, said peptide comprising a cell targeting motif sequence conjugated to the peptide via a spacer sequence wherein the cell targeting motif is the metastatic prostate cancer targeting peptide TMTP-1 (NWRQ, SEQ ID No. 12) and the spacer is an alpha helical spacer comprising from 1 to 4 repeats of the sequence EAAAK (SEQ ID No. 13).
20. The cell delivery system according to claim 8, said peptide comprising a cell targeting motif sequence conjugated to the peptide via a spacer sequence wherein the cell targeting motif is the metastatic prostate cancer targeting peptide TMTP-1 (NWRQ, SEQ ID No. 12) and the spacer is an alpha helical spacer.
21. A method of improving the bioavailability of a phosphate based drug to an individual, comprising the administration of the cell delivery system according to claim 8, when complexed with a phosphate based drug, to an individual in need thereof.
22. A method of improving the cellular uptake of gold to an individual in need thereof comprising the administration of the cell delivery system according to claim 8 when complexed with gold, to an individual.
Description
(1) The present invention will now be described with reference to the following non-limiting figures and examples.
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27) Following preparation of RALA/pORF-mIL4 nanoparticles N:P 10 they were characterised over a range of temperatures (4-37° C.) and following incubation at room temperature for up to 6 h using the Malvern Zetasizer NanoZS with DTS software. The measurements are reported as mean±SEM, (n=3).
(28)
(29)
(30)
(31)
(32)
(33)
(34)
(35)
(36)
(37)
(38)
(39)
(40)
(41)
(42)
(43)
(44)
(45)
(46)
(47)
(48)
(49)
(50)
(51)
(52)
(53)
(54)
(55)
(56)
(57)
EXAMPLES
Example 1: Generation of RALA Peptide
(58) The following peptide (called “RALA” herein) was synthesised commercially in accordance with conventional techniques with the amino acid sequence
(59) TABLE-US-00007 WEARLARALARALARHLARALARALRACEA
RALA arrives in a lyophilised form and is reconstituted with molecular grade water to a desired concentration, aliquotted out and stored at −20° C. until further use. An aliquot is then taken as needed and defrosted on ice.
Example 2: Formation and In-Vitro/In-Vivo Testing of RALA/SIRNA Nanoparticles
(60) Materials and Methods
(61) Calculation of N:P Ratio
(62) DNA was complexed with either the RALA peptide at various N:P ratios (the molar ratio of positively charged nitrogen atoms to negatively charged phosphates in DNA). As the number of positive side-groups in a protein side chain depends upon the sequence, different proteins will have differing numbers of positive charges per unit mass. In order to calculate this, the following equation was used:
NP=M.sub.protein/M.sub.DNAC.sub.NP
(63) Where M protein is the mass of a protein, M DNA is the mass of DNA and C NP is the N:P constant. The N:P constant is the ratio of the protein's side chain positive charge density to the DNAs backbone density, with the charge density being the charge of a substance divided by its molecular mass. For the protein, lysine, arginine and histidine side groups are counted. For the DNA the average mass of one single base pair, and the charge of the phosphate group are used. For RALA an N:P ratio of 1 is 1.45 μg of RALA:1 μg of DNA.
(64) Formation of the Nanoparticles
(65) The DNA/siRNA was diluted in molecular grade water to 200 μg/ml. 1 μg of DNA was added to a 1.5 ml eppendorf centrifuge tube. For 1 μg of DNA the final volume was 50 μl. The appropriate volume of protein to use to make the desired N:P ratio was added to a separate tube and the volume made up to 50 μl with molecular grade water. The 50 μl solution containing the protein was added to the 50 μl containing the DNA. The molecular grade water was added to the DNA before the protein. The tube was flicked five times in order to mix the content. The complexes were allowed to incubate for 30 minutes at room temperature prior to use. The results are shown in
(66) Gel Retardation Assay
(67) RALA/DNA complexes were prepared at N:P ratios 1-15. Following incubation at room temperature for 30 minutes, 30 μL of the samples (corresponding to 0.6 μg of DNA) were electrophoresed through a 1% agarose gel containing 0.5 μg/mL ethidium bromide (EtBr) (Sigma, UK) to visualize DNA. A current of 80 V was applied for 1 h and the gel imaged using a Multispectrum Bioimaging System (UVP, UK). The purpose of this assay is to determine which N:P ratio/s neutralise the DNA. The assay works upon the principle that when complexes are formed with an excess positive charge DNA remains in the wells or migrates up the gel, hence, no DNA band will be visible following gel electrophoresis. However, DNA alone or complexed to give a net negative charge will migrate down the gel (
(68) Nanoparticle Size and Charge Analysis
(69) In order to obtain particle size and charge distributions the mean hydrodynamic particle size measurements RALA complexes were performed using Dynamic Light Scattering (DLS). Dynamic Light Scattering is based upon the principle that when particles are illuminated with a laser, due to Brownian motion there will be scattering of the light. The intensity of the scattered light fluctuates as a result of this Brownian motion caused by bombardment of the particles by solvent molecules. A correlation curve reflecting the decay rate is generated based on fluctuations of the scattered light where a slower correlation decay rate represents a slower moving particle. Based on the Stokes-Einstein equation larger particles move more slowly and, thus, the correlation function can be used to determine the size distribution of the particles. dynamic light scattering (DLS) was used.
(70) Surface charge measurements of the RALA nanoparticles were determined by Laser Doppler Velocimetry. The zeta potential of the particles was measured using disposable foltable zeta cuvettes. Zeta cuvettes for the measurement of zeta potential were first washed with 70% ethanol, followed by two rinses with double distilled H2O prior to loading the sample. Enough diluted sample used for size measurement was used for determination of zeta potential.
(71) The nanoparticles were made up at an appropriate range of N:P ratios with at least using 2 μg of DNA in each sample. Nanoparticles were analysed using either and analysis was completed on either the Zetasizer-HS3000 (Malvern Instruments) or the Zetasizer-Nano instrument with DTS software (Malvern Instruments, UK). Zetasizer-Nano (Malvern Instruments) (
(72) Incubation Stability Study of RALA Nanoparticles
(73) This assay is designed to illustrate the stability of RALA complexes to indicate the optimal time period for nanoparticle formation. Following incubation at room temperature for 30 min the mean hydrodynamic size and zeta potential were measured using the Malvern Zetasizer NanoZS with DTS software at 15 or 30 min intervals over a period of 360 min. Size and zeta potential are reported as mean±SEM, n=3, where n represents the number of independent batches prepared for measurement (
(74) Temperature Stability Study of RALA Complexes
(75) This assay determines the stability of the nanoparticles over a range of temperatures. Following preparation of the nanoparticles by incubation at room temperature for 30 min the mean hydrodynamic size and zeta potential were measured over a temperature range of 4-37° C. in 4° C. intervals using the Malvern Zetasizer NanoZS with DTS software. The sample was allowed to equilibrate at each temperature for 120 sec before measurements were taken in triplicate. Results are reported as mean±SEM, n=3, where n represents the number of independent batches prepared for measurement (
(76) Serum Stability Assay
(77) In order to determine the stability of the RALA nanoparticles when exposed to serum the following procedure was carried out. Six replicates of the complexes at NP ratios 5, 10 and 15 were made. Each N:P ratio was split into 3 aliquots or in the case of RALA 18 aliquots. 10% foetal calf serum was added to 12 of the aliquots. The 18 aliquots were incubated at 37° C. Every 55 min SDS (sodium dodecyl sulphate (Sigma, UK)) was added to one of aliquots containing serum for each N:P ratio which were then incubated for a further 5 min. For RALA the stability was assessed over a 6 h time course. Loading dye (Ficoll (Sigma, UK), Tris-HCl, bromophenol blue (Sigma, UK) in ddH2O) was added to all the aliquots prior to loading onto an ethidium bromide prestained 0.8% agarose-TAE gel. A current of 80V was applied for 1 h and the gel was visualised using a Multispectrum Bioimaging System (UVP, UK). (
(78) Transmission Electron Microscopy
(79) In an attempt to confirm the results obtained by DLS and obtain additional information about the structure of the nanoparticles Transmission Electron Microscopy was employed. The RALA complexes were prepared as pert or standard conditions and 5 μl was pipetted onto formvar coated copper grids (Agar Scientific, UK) and allowed to air dry overnight. Subsequently samples were stained with 5% aqueous 5% uranyl acetate for 5 minutes and allowed to dry overnight before visualisation. The nanoparticles were imaged using JEOL 100CXII transmission electron microscope at an accelerating voltage of 80 kV (
(80) Freeze Drying of the Nanoparticles
(81) 700 μl of RALA-pEGFP-N1 nanoparticles were subject to freezing for 1 h at −40° C. This was followed by primary drying at −40° C. and 60 mTorr for 24 h. This was followed by the secondary drying program; 3 h at −35° C. and 120 mTorr, 3 h at −30° C. and 190 mTorr, 3 h at −25° C. and 190 mTorr and 6 h at 20° C. (
(82) Transfection of ZR-75-1 & PC-3 Cells in 96 Well Plates with the RALA Nanoparticles
(83) In order to test the RALA in vitro, small scale transfections were performed carried out. 5×104 cells were seeded onto each well of a 96 well plate and the cells incubated under with complete medium standard conditions for 48 hours. The medium was subsequently removed from the plates and 100 μl of transfection medium (Optimem Invitrogen, UK) was added to each well. Cells were incubated for 2 hours at 37° C. and 5% CO2 standard conditions. In the meanwhile complexes were made up using 1 μg of plasmid DNA with the RALA vector and added to the cells when the two hours had passed. 100 μl of the each N:P ratio were added to each well of the cells. Cells were then incubated for a further 4 hours under standard conditions and the medium with RPMI-1640 supplemented with +10% FCS. (
(84) Flow Cytometry to Quantify Fluorescent Intensity
(85) ZR-75-1 & PC-3 cells that were transfected with RALA/pEGFP-N1 complexes were trypsinised and washed twice with 2% formaldehyde in phosphate buffered saline. The expression of green fluorescent protein was measured by flow cytometry using FACS calibur system (BD Bioscience, UK). The data was analysed using the Flo-Jo software program and fluorescent intensity is reported at 4% gating. (
(86) Cell Proliferation Assay
(87) Cell viability was evaluated by manual counting of the viable adherent cells using a haemocytometer as described in. PC-3 prostate cancer cells were seeded in a 96-well flat-bottom tissue culture plate at a density of 1×104 cells per well and incubated in complete culture medium for 24 h. Two hours prior to transfection the cells were conditioned in OptiMEM serum-free medium (Invitrogen, UK) optimised for transfection. Cells were treated with solutions of BP to achieve a final exposure concentration of 5 μM to 1 mM. RALA/BP nanoparticles were prepared using a mass ratio of 10:1 such that the final concentration of BP per well was in the range 5 μM to 75 μM. Cells were incubated at 37° C. with 5% CO2 for 6 h before medium was replaced with completed culture medium and left to incubate for 72 h. Following incubation the cells were trypsinised and counted. Cell viability was expressed as a percentage of the untreated control where the untreated control is considered to be 100% viable. Dose-response curves were obtained for free BP and RALA/BP allowing determination of EC50 values for each. EC50 values refer to the concentration that induces a response halfway between the baseline and the maximum plateau obtained (
(88) WST-1 Cell Viability Assay
(89) The WST-1 assay is a colorimetric assay that can analyse the number of viable cells present and hence, indicate the toxicity of complexes added to cells in vitro. The assay is based on the cleavage of tetrazolium salts that are added to the culture medium. The stable tetrazolium salt WST-1 is cleaved to a soluble formazan by a cellular mechanism that occurs primarily at the cell surface. This WST-1 cleavage is dependent on the glycolytic production of NAD(P)H in viable cells, therefore, the amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture.
(90) Cells were transfected and the complete medium was discarded at a range of time points and replaced with 100 μL Opti-MEM with 10% WST-1 reagent (Roche, UK). Cells were incubated for 2 h under standard cell culture conditions. Subsequently the plates were shaken for 1 min and absorbance measured at 450 nm on an EL808 96-well plate reader (Biotek, USA). The measured absorbance values are expressed as a percentage of the control where the control is defined as 100% viable (
(91) Intradermal Tumour Model in BALB-C SCID Mice
(92) ZR-75-1 or PC-3 cells were trypsinised until they had detached and 8 ml of medium was added per flask. The cell suspension was transferred into 20 ml universal tubes. The cells were and centrifuged for 5 minutes at 80 g. Cells were resuspended in RPMI+10% FCS and counted using a Coulter Counter (Beckman Coulter, UK). Cells were subsequently centrifuged as before, and resuspended at 108 cells per ml in PBS before being diluted 1:in 1 in matrigel (BD Biosciences, UK). The matrigel cell suspension was loaded into syringes and kept on ice until implantation. Matrigel was only required for the ZR-75-1 cells. Balb-C SCID mice were anaesthetised with isofluorane (Abbott, UK) and the rear dorsum was shaved. Subsequently the skin on the rear dorsum was pinched between forefinger and thumb and 5×106 cells (100 μl) were injected intradermally using with a 26G needle (BD Biosciences, UK) at the prepared site. Mice were observed while recovering from the anaesthesia and then subsequently returned to their box (
(93) Tumour Size Measurements
(94) The length (L), width (W) and depth (D) of the tumour was measured using vernier with calipers. Subsequently the volume of the tumour was estimated by using the equation, V=πLWD/6, an approximation of V=4/3πr3.
(95) Intra-Tumoural Injections
(96) Mice were anaesthetised with isofluorane and a 26G needle (BD Biosciences, UK) was inserted bevel side down into the tumour. 100 μl of the nanoparticle treatment was injected slowly before rotating the needle and removing very slowly. For the multiple dose regimen used in this study a ‘round the clock’ system of injections was used. Recovery of mice from anaesthesia was monitored (47,52).
(97) Intra-Venous Injections
(98) Mice were placed into a heat box at 36° C. for 5 minutes or until both of the tail veins were clearly visible. They were then moved into a heavy brass restrainer and injected with 50-100 μl of treatment into the tail vein with an insulin syringe (BD Biosciences, UK) equipped with 28G needle. Mice were then replaced into the cage and monitored for signs of suffering associated with the injection. Mice found to be suffering or dying were euthanized by a schedule one protocol. (
(99) Harvesting Blood Via Cardiac Puncture and Collection of Serum
(100) For the harvesting of blood and intraperitoneal macrophages, cervical dislocation was the preferred method of euthanasia. Cardiac puncture was performed using a 21G gauge needle (BD Biosciences, UK). The needle was placed horizontally slightly to the left side of the sternum to go up through the diaphragm. The needle was then withdrawn very slowly until ˜500 μl of blood was collected and placed in an eppendorf. The eppendorf was then stored at room temperature with an open lid to facilitate coagulation. After 30 min the eppendorfs were centrifuged at 2000 rpm for 10 min. The supernatant containing the serum was carefully decanted and placed into a clean eppendorf and stored at −20° C. until further use. When harvesting intraperitoneal macrophages an incision was made and the peritoneal cavity was flushed out with 30% sucrose (Sigma, UK) solution. The macrophages were stored at 4° C. until they could be cultured (
(101) Western Blots with In Vitro and In Vivo Samples
(102) Organs were homogenised and lysed in RIPA overnight. The samples were centrifuged at 5000 g for 10 minutes and the supernatant transferred to a fresh eppendorf tube. The lysate was diluted 1:2 in laemmli buffer, boiled for 10 minutes and loaded onto a Bis-Tris gel. Cells were put directly into laemmli buffer. The gel was run at 120V till the dye reached the bottom. The gel was and transferred into a western cassette. The protein was subsequently transferred for 2.5 hours at 25V onto a nitrocellulose membrane (Amersham, Biosciences, UK). Protein transfer was visualised by staining with Ponceau stain (Sigma, UK). The membrane was then subsequently incubated with primary antibody in blocking solution (PBS (Invitrogen, UK), 0.1% Tween (Sigma, UK), Skimmed milk (Merck, Germany)). Subsequently the membrane was then rinsed twice within Tween-PBS and once within PBS before being incubated in secondary antibody for 1.5 hours. The membrane was then was rinsed again, twice with Tween-PBS and once within PBS before the application of Immobilon reagent (Millipore, UK). Western blots were quantified using imageJ software (
(103) Vector Neutralisation Assay
(104) Female C57/BL6 mice (5-6 weeks old) were treated with one of; PBS (control) RALA alone DNA alone (CMV/GFP) RALA/DNA nanoparticles
(105) Mice receiving DNA received 10 μg total. Nanoparticles were formulated with an N:P ratio of 10. Mice receiving RALA alone received an amount of vector equivalent to that received in the RALA/DNA group. Treatments were administered by tail vein injection performed over a three week period. There was 15 mice per treatment group, with 5 mice per time point.
(106) All animals received the relevant treatment on Day 0. Following 7 days, five mice from each group were sacrificed and blood from each will be isolated by cardiac puncture. Serum was isolated, serum from the five mice per group was pooled, heat-inactivated at 56° C. for 30-60 min, and serially diluted in Opti-MEM to produce serum concentrations of 10% v/v, 1% v/v and 0.1% v/v, plus a 0% control.
(107) To these serum dilutions, fresh RALA/DNA nanoparticles (as above) were added at a DNA concentration of 1 μg/200 μl (the standard concentration for RALA/DNA transfection in 96 well plate format), and incubated at 37° C. for 1 h. This pre-incubated mix was then transferred to ZR-75-1 breast cancer cells previously seeded in 96 well plates (104 cells/well) on Day 6, and transfection was performed in the usual manner. Transfection of the GFP construct was assessed by FACS analysis after 24 h.
(108) On Day 7, the remaining 10 mice received a second administration of the appropriate treatment. On Day 14, five mice left the experiment and were treated as above, while the remaining five mice per group received a final administration of the appropriate treatment, and on Day 21, followed by the previously outlined treatment (
(109) Enzyme-Linked Immunosorbent Assay
(110) These assays were performed on the serum collected from immunocompetent C57/BL6 mice following either 1, 2, or 3 intravenous injection with the RALA/pEGFP-N1 nanoparticles. IgG, IgM, II-12, IL-6, and TNF-β, ELISAs were performed using the ENZO ELISA Kits in accordance with the recommended protocol (
(111) For the neutralising antibody ELISA the following method applied;
(112) Nunc Maxisorp ELISA plates were coated with RALA-pEGFP nanoparticles equivalent to 1 μg DNA per well. The wells were subsequently blocked with PBS/5% BSA. Wells were probed for 1 h with sera from mice diluted (1:500) in PBS/0.5% BSA at room temperature. (NB the sera came from the mice treated in the vector neutralisaiton assay). The wells were washed with PBS/0.5% Tween 20 and then probed for 30 min with HRP-conjugated anti-mouse secondary antibody. Wells were then washed again and probed with TMB substrate for 30 min. Colour development was measured at 450 nm with a reference wavelength of 550 nm (
(113) Confocal Microscopy
(114) 5000 ZR-75-1 breast cancer or PC-3 prostate cancer cells were grown on cover slips and transfected with Cy3 labelled RALA/pEGFP (lacks the promoter contained in the construct used in the neutralisation assay) or fluorescent siRNA. Confocal microscopy was used to determine subcellular localisation of RALA/Cy3-pEGFP nanoparticles (
(115) Gold Nanoparticle Experiment
(116) 5 nm phosphorylated gold nanoparticles were incubated with RALA peptide at a ratio of approximately 1:10 for 30 mins before being added to MDA-MB-231 breast cancer cells for 24 hours. The MDA-MB-231 cells (5000) had been seeded onto a coverslip. After 24 h the cells were fixed with 50% methanol and 50% acetone and sent to Cytoviva (Auburn, Ala.) for imaging (
(117) Greiss Test
(118) Cells seeded in multiwell plates (6 or 24 well) were transfected with various amounts of pDNA (CMV/iNOS or hOC/iNOS) complexed with RALA at N:P 10 for 6 h, following which, transfection complexes were removed, and cells returned to normal growth medium (Minimum Essential Medium—MEM). After 48 h, 70 μl aliquots of conditioned MEM were assayed for their total nitrate (an indirect indicator of nitric oxide content) content using a Nitric Oxide Quantitation kit (Active Motif) following the manufacturer's instructions. A standard curve (using 0-35 μM sodium nitrate) was constructed and used to quantify nitrate content in sample wells of the assay plate. After incubation of standards and unknown samples with nitrate reductase and co-factors, Greiss reagents A and B were added to wells, and after a 20 min incubation to allow colour development, the absorbance of each well at 540 nm was determined (
(119) Clonogenic Assay
(120) PC-3s grown in T25 tissue culture flasks were starved of serum by Opti-MEM incubation for 2 h before transfection with 10 μg of pDNA (CMV/iNOS, hOC/iNOS or CMV/GFP) for 6 h. Following transfection, media were replaced with MEM, and the cells incubated overnight. The next day, cells were trypsinised, resuspended in growth medium, enumerated, and plated in triplicate into 6 well plates (200 or 500 cells per well). Plates were incubated for 14 days to allow clonogenic growth, following which, medium was aspirated, colonies were stained with crystal violet and counted manually. Percentage cell survival was calculated by comparison with untransfected cells (
(121) Intracardiac Metastases Model
(122) Female Balb/c SCID mice (5-8 weeks old) were inoculated via the left cardiac ventricle with 2×105 MDA-MB-231-luc2 breast cancer cells that express firefly luciferase. Mice then received an intraperitoneal injection of 200 μl D-luciferin (15 mg/ml) and were imaged (following 10 min) using IVIS imaging; successful left ventricular delivery was confirmed by whole body luminescence immediately following intracardiac delivery. Mice possessing luminescence limited to the thoracic cavity were sacrificed at this point. Remaining successfully inoculated mice were randomly assigned to one of four treatment groups (water, RALA only, RALA-CMV/iNOS or RALA-hOC/iNOS), and received five treatments twice weekly commencing two days post inoculation. Gene therapy mice received 10 μg pDNA complexed with RALA at N:P 10, RALA only mice received the corresponding amount of RALA dissolved with water; treatments were of 100 μl, and were delivered via the tail vein. Mice were routinely imaged twice weekly as described above, were observed daily by experienced animal husbandry experts, and body mass was monitored as an indicator of general health. A loss of 20% of original body mass was considered indicative of poor health of the mice, and this combined with a moribund appearance was determined to be a humane experimental end point (
(123) Effect of Runx2 Knockdown on Cell Proliferation
(124) The effects of Runx2 knockdown on cell proliferation were evaluated at different time-points following transfection with RALA/Runx2 siRNA nanoparticles. Nanoparticles were prepared such that the final concentration of Runx2 siRNA was 100 nM and based on a N:P ratio of 12. Two Silencer Select Runx2 siRNAs were used and a Silencer Select non-coding siRNA (Invitrogen, UK). Cells were serum starved for 2 h prior to transfection. Transfections were carried out with both RALA peptide and Oligofectamine for a duration of 4 h in serum-free RPMI 1640 before RPMI 1640 containing 30% FCS was added to achieve a final FCS concentration of 10%. After 24, 48 and 72 h cells were detached using 2× trypsin and subsequently neutralised with RPMI 1640 containing 10% FCS. Cells were counted manually using a haemocytometer as described in 3.2.11.2 and the cell viability determined based on the assumption of a 100% viability of the untreated cells. Results are reported as mean±SEM, n=3, where n represents the number of independent batches prepared for analysis (
(125) Western Blotting for Runx2 Protein
(126) To assess the ability of RALA/Runx2 siRNA nanoparticles to successfully inhibit Runx2 protein expression a range of siRNA concentrations and time-points following transfection were evaluated by Western blotting. PC-3 prostate cancer cells were seeded at a density of 150,000 cells per well in a 12-well plate. Transfections were initially carried out with various amounts of two types of Silencer Select Runx2 siRNA and Silencer Select non-targeting control siRNA such that the final siRNA concentration in the well was 50, 100 or 200 nM. Transfection was for 4 h followed by 48 h incubation. Following optimisation of the concentration the optimal time following transfection was determined using 100 nM concentrations. Cells were washed with ice-cold tris buffered saline (TBS) and lysed in a direct lysis buffer supplemented with MG-132 (Calbiochem, UK) and protease inhibitor cocktail (Roche, UK) (Appendix 1). Lysed samples were stored at −20° C. until required.
(127) Samples were run on 8% acrylamide gels at 100 V for 15 min followed by 150 V until the dye front reached the bottom of the gel in a tris-glycine running buffer. Subsequently the protein was transferred to PVDF membranes at 200 mA for 90 min in a tris-glycine transfer buffer. Membranes were blocked for up to 1 h in 2% blocking solution before leaving in primary antibody overnight at 4° C. with rocking. Runx2 primary antibody (MBL International, Woburn, Mass.) was used at a concentration of 1:200 and 8-actin (Abcam, UK) at a concentration of 1:5000. Membranes were washed in TBS-tween (TBS-T) for 30 min before applying anti-mouse secondary antibody at 1:5000 for 1 h at room temperature. Membranes were washed vigorously in TBS-T for 30 min before developing. The chemiluminescent used for Runx2 protein was Thermo Scientific SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, Mass.) and for β-actin Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, Mass.) (
(128) Studies with the RAT Peptide
(129) RAT was synthesised from a commercial company and is a fusogenic, consisting of RALA with an alphahelical concatemeric spacer, (EAAAK)4, and the TMTP1 (NVVRQ) metastatic prostate cancer targeting peptide (
(130) RALA-PEG5k
(131) A pegylated version of RALA has been synthesised (
(132) Composite RALA Nanoparticles
(133) RALA nanoparticles were prepared using desalted peptide in MOPS buffer at 50° C. to give a concentration of 50 μg/ml of DNA. PLGA and a series of PLA-PEG block copolymers were synthesized with various PEG chain length and LA/EG ratio (PLA10-PEG2; PLA25-PEG5; PLA50-PEG5) and formulated into composite nanoparticles (diameter<200 n m and PDI<0.2000) containing the RNPs. 100 μl of RALA nanoparticles was added to 0.5 ml 4% w/v copolymeric polymeric solution in dichloromethane under vortex and probe sonicated (120 Sonic Dismembrator with 3 mm probe, Fisher Scientific, USA) for 60 seconds at 50% of amplitude. This water-in-oil (w/o) emulsion was added to 2.5 ml of 5% w/v PVA solution in distilled water under vortex and probe sonicated as before in an ice bath for 2 minutes. The resultant emulsion was stirred overnight to form the composite nanoparticles. These were collected by centrifugation at 30,000 g for 30 min (3K30, Sigma Centrifuge, UK) and washed twice with distilled water, before suspending in 1 ml 5% w/v trehalose in water and were freeze-dried (Advantage, VirTis, Gardiner, N.Y., USA). TEM (JEOL JEM1400 transmission electron microscope at an accelerating voltage of 80 kV) was performed by loading samples onto a copper grid (Formvar/Carbon 200 mesh, Agar scientific). Osmium tetraoxide was incorporated by adding it to the organic phase during preparation of the composite nanoparticles.
(134) Results
(135) Particle Characterisation
(136) As shown in
(137) Particle formation between DNA and RALA was studied by gel retardation assays and dynamic light scattering. It was found that RALA fully condensed DNA at N:P ratios above 4 (
(138) Additionally, serum stability of particles at N:Ps of 5, 10 and 15 showed that the nanoparticles are stable in the presence of 10% serum and dissociate in 1% SDS revealing that the integrity of the DNA remains intact (
(139) In Vitro Transfection Efficacy & Cytotoxicity
(140) ZR-75-1 cells were transfected with RALA/pEGFP-N1 nanoparticles. Epi-fluorescence microscopy showed a high transfection efficacy of ZR-75-1 cells, when transfected with RALA/pEGFP at N:P of 10 with and without chloroquine. Chloroquine is a known endosomal disrupter and will increase transfection if the nanoparticles are inefficient endosome disrupters. At N:P 10 this is clearly not the case. Flow cytometry was then used to further analyse the effect of N:P on transfection efficacy and revealed an optimal transfection efficacy of around 30% between N:P ratios 8-12. More importantly though the WST-1 cell viability assay revealed minimal toxicity of the nanoparticles over a range of N:P ratios. Cell viability was 90% for N:P 4 and 80% at N:P 10. Indeed when cellular proliferation was examined there was significant difference between lipofectamine 2000 and RALA/pEGFP-N1 transfected cells (
(141) To determine if RALA/pEGFP-N1 nanoparticles are significantly more efficient at eliciting cellular transfection in comparison to KALA/pEGFP-N1 nanoparticles a transfection experiment with both peptide-based nanoparticles was carried out in parallel (
(142) Confocal microscopy also confirmed successful transfection with a time course revealing diffuse pattern of distribution of nanoparticles that focus into distinct foci with increasing duration of transfection (
(143) Lyophylisation of RALA
(144) As RALA/pEGFP-N1 nanoparticles transfect cells efficiently and are non-toxic, it was decided to use these nanoparticles as a model of a potentially therapeutic peptide based polyplex. It is well know that a major problem with gene therapy protocols is storage as both peptide and DNA degrade if stored in aqueous solutions at room temperature for prolonged periods of time. As such, the nanoparticles were lyophylised with a range of concentrations of trehalose as a lyoprotectant. Transfections, as well as serum stability assays were performed before and after freeze-drying. Serum stability assays were performed on all formulations up to 6 h. All formulations were found to be as stable upon incubation with 10% serum as the fresh particles without trehalose (
(145) Overall these results highlight the stability of RALA/pEGFP-N1 nanoparticles as well as the ease with which dried formulations can be stored, even without lyoprotection. These data indicate that the RALA could be lyophilised, stored and reconstituted prior to administration without losing activity.
(146) Transfection Efficacy & Immunogenicity of RALA In Vivo
(147) As RALA has proven highly effective in vitro, the next logical step would be to test its transfection efficacy and distribution and most importantly, bio-compatability in vivo. As such, ZR-75-1 tumour bearing BALB/C-SCID mice were injected intravenously with 50 μl of N:P 10 RALA/pEGFP-N1 or RALA/phOCMetLuc nanoparticles carrying a total of 10 μg of plasmid DNA per dose. Western blots showed transfection in all organs with the pEGFP-N1 carrying nanoparticles and in the tumour, surrounding tissue and liver with phOCMetLuc nanoparticles (
(148) In order to determine whether the RALA based nanoparticles would be safe for repeated administration, immunocompetent C57/BL6 mice were treated once a week with either 50 μl of PBS, PEI, RALA, pEGFP-N1, PEI/pEGFP-N1 or RALA/pEGFP-N1 for 3 weeks. In each instance the dose of plasmid DNA delivered was 10 μg. Blood was collected via cardiac puncture and ELISA's were performed for IgGs, IgMs, TNFα, IL6 and IL1β, alongside a Greiss test for increased nitric oxide concentrations. No morbidity or visible immune response was seen upon inspection of the live animals. ELISAs for interleukins yielded no statistically significant differences between groups of treatments (
(149) Furthermore multiple injections of the RALA nanoparticles did not evoke neutralising antibodies that would prevent RALA from delivering its payload. FACS analysis of PC3 and ZR-75-1 cells indicated that transfection of both cell types was hampered by the presence of 10% serum, but this occurred with the FBS controls as well eliminating the activation of an immune response (
(150) Systemic Delivery of RALA/iNOS Nanoparticles
(151) Transfection of PC-3 and MDA-MB-231 with plasmid iNOS constructs complexed with RALA evoked nitric oxide production (as determined by total nitrate content of growth media—an indirect method of nitric oxide quantification). PC-3s and MDA MB-231s transfected with the inducible hOC/iNOS plasmid produced significantly more nitrates than were present in control (P=0.038 and 0.048 respectively), and those transfected with the constitutively active CMV/iNOS also produced levels of nitrates considerably higher than seen in control. Nitrate content of media of cells transfected with green fluorescent protein constructs under the control of the same promoters were consistent with control (
(152) Transfection of PC-3s with hOC/iNOS complexed with RALA prior to clonogenic assay resulted in significantly lower clonogenic survival compared to control (P=0.004). Transfection of the same cells with CMV/iNOS resulted in a roughly similar loss of clonogenic survival (0.69±0.08 vs 0.61±0.03), while transfection with CMV/GFP did not affect clonogenic survival of PC-3s (surviving fraction of 1.01±0.11) (
(153) Metastatic deposits were established in female BALB/c SCID mice by inoculation with 2×105 MDA-MB-231-D3H1 that express luciferase via the left ventricle of the heart. Metastatic development was monitored routinely by IVIS imaging of bioluminescence (
(154) Delivery of RALA/siRUNX2 as a Therapeutic
(155) To confirm that Runx2 protein expression could successfully be knocked down using the RALA, PC-3 prostate cancer cells were transfected and the cell lysate collected for Western blotting. Two types of Runx2 siRNA were used as well as a non-targeting scrambled siRNA. Furthermore, Oligofectamine was used as a positive control for comparison. Initially the concentration of siRNA required to achieve knockdown was assessed followed by the optimal incubation time post-transfection. Densitometry of the Western blots using Image J software enabled the degree of knockdown of protein expression to be quantified by assuming the scrambled control siRNA results in 0% knockdown.
(156)
(157) RALA peptide was able to achieve comparable levels of knockdown to the commercial RNA transfection reagent, Oligofectamine. Analysis of the transfection profile of RALA and Oligofectamine using fluorescent siRNA showed a peak in transfection immediately after transfection with RALA but it took 24 h to reach a peak with Oligofectamine.
(158) To determine the effects of Runx2 knockdown on prostate cancer cell proliferation, PC-3 prostate cancer cells were transfected with 100 nM Runx2_1, Runx2_2 or non-targeting scrambled siRNA using RALA or Oligofectamine as a positive control. Where RALA was used nanoparticles were prepared at N:P 12 and Oligofectamine was used as per the manufacturer's guidelines. Cells were trypsinised and counted using a haemocytometer at 24, 48 and 72 h following the 4 h transfection. Untreated cells were assumed to have 100% viability and the percentage viability for all other treatments was based on this.
(159) Cell viability was significantly lower with Runx2_1 compared to Runx2_2 24 h following transfection with RALA peptide (p=0.0376). However, no significant difference between the two siRNAs is seen at any other timepoint or following delivery using Oligofectamine (p>0.05) as determined by two-way ANOVA. Furthermore, there is no significant difference in cell viability following transfection of Runx2_1 and Runx2_2 across the timepoints studied up to 72 h (p>0.05) when determined by two-way ANOVA. RALA/Runx2_1 siRNA nanoparticles resulted in a significant reduction in cell viability when compared to RALA/scrambled siRNA nanoparticles at each of the 24, 48 and 72 h timepoints evaluated (p<0.001, 0.05 and 0.01 respectively). Similar results were found with RALA/Runx2 siRNA nanoparticles (p<0.01, 0.01 and 0.001 respectively). These results were consistent with the positive control, Oligofectamine, which also resulted in a significant decrease in cell viability compared to the scrambled control with Runx2_1 (p<0.001, 0.01 and 0.001 at 24, 48 and 72 h respectively) and Runx2_2 (p<0.01, 0.05 and 0.01 at 24, 48 and 72 h respectively). Overall, knockdown of Runx2 protein expression results in a reduction in cell viability of approximately 30% over 72 h (
(160) Tumours were grown on the rear dorsum of BALB-C SCID mice until the volume reached approximately 150 mm3 before intratumoural treatment with either RALA/Runx2 siRNA nanoparticles, Runx2 siRNA only or RALA/scrambled siRNA nanoparticles commenced. Runx2_1 and Runx2_2 siRNA were pooled for the purposes of in vivo analysis as neither was found to be significantly better in achieving Runx2 knockdown. Dosing was once weekly until tumour quadrupling defined the endpoint of the experiment. Control tumours grew rapidly with all tumours quadrupling in volume within 16 days of the start of treatment (average 15 days). RALA/scrambled siRNA nanoparticle treatment mice follow a similar rate of growth as the untreated. The rate of growth is also similar for Runx2 siRNA treated mice until after the second treatment; following this the tumours grow at a slower rate than the untreated and RALA/scrambled siRNA groups. In mice treated with RALA/Runx2 siRNA nanoparticles, tumours grow at a slower rate than all other groups until the point of tumour volume quadrupling (
(161) Delivery of RALA/BP as a Therapeutic
(162) In order to assess the effectiveness of RALA as a delivery agent for optimisation of the antitumour effects of BPs, PC-3 prostate cancer cells were either treated with free BP or transfected with RALA/BP nanoparticles at a range of concentrations for 6 h and then incubated for 72 h before evaluating cell viability. Cell viability was analysed by cell counting using a haemocytometer. EC50 values were determined using the dose-response curves generated from this cell viability data. The EC50 of alendronate was reduced from 100.3 μM to 17.6 μM when delivered in a RALA nanoparticle, a potentiation factor of 5.7 (
(163) Tumours were grown on the rear dorsum of BALB-C SCID mice until the volume reached approximately 100 mm3 before intratumoural treatment with RALA/alendronate, alendronate or RALA commenced. Dosing was thrice weekly until tumour quadrupling defined the endpoint of the experiment. It can be seen clearly that RALA only had no significant effect on tumour growth (p=0.0792) while alendronate and RALA/alendronate show high statistical significance when compared to the untreated control (p<0.0001 and p=0.0004 respectively) (
(164) RAT Results
(165) RAT was synthesized (
(166) A serum incubation study was used to determine if RAT/pEGFP-N1 nanoparticles were stable over a 6 h time period with and without the presence of foetal calf serum (
(167) The specificity of the RAT peptide was assessed using a targeting inhibition study (
(168) TEM also confirmed the presence of the RALA nanoparticles inside the composite nanoparticles (
(169) In summary, the results presented show that RALA is efficient, stable, safe and a viable delivery vehicle for iNOS DNA, RUNX2 siRNA and bisphosphonate anti-cancer therapeutics.
CONCLUSION
(170) The physical properties of the RALA/pEGFP-N1 nanoparticles have been analysed and their efficacy as a transfection agent demonstrated both in vitro and in vivo. RALA was found to form stable complexes with pEGFP-N1 and facilitate the transfection of ZR-75-1 cells. Gel retardations show that complexes are formed at N:P ratios as low N:P 1, but full complexation is not seen until N:P 4, which is comparable with KALA and ppTG peptides [Rittner et al. 2002]. The RALA/pEGFP-N1 complexes cannot be defined as nanoparticles until N:P 4, as their size at N:P ratios 2 and 3 was in the micrometer range. At ratios of N:P 4 and above, RALA forms nanoparticles with pEGFP-N1 with a positive charge of 30 mV. This is in agreement with the counter-ion condensation theory, which states that particle sizes of charged complexes should be lower than those of uncharged particles, as electrostatic repulsion should prevent aggregation [de Smedt et al. 2000, Bagwe et al. 2006].
(171) Given that at the N:P ratios which yield the highest transfection efficacy, the particles have a positive surface charge and a mean diameter below 100 nm, it is possible that they bind to the negatively charged cell surface proteoglycans non-specifically and are subsequently taken up into the endosomes.
(172) With respect to transfection efficiency, the use of arginine in the RALA peptide has two distinct advantages; firstly arginine has consistently been shown to be the optimal amino acid for condensing DNA with arginine rich sequences binding in milliseconds (Murray et al 2001). Secondly arginine rich sequences based on the Rev sequence have the capacity to actively transport DNA into the nucleus of cells via the importin pathway (Malim et al 1989). This gives RALA a distinct advantage over conventional peptide delivery systems.
(173) We have also shown that the RALA/pEGFP-N1 nanoparticles are not strongly cytotoxic, causing only a 20% reduction in cell viability in transfected cell monolayers. Perhaps the most important result is the confirmation of in vivo activity of the nanoparticles following systemic administration. High levels of delivery to the lungs were seen when a plasmid expressing luciferase was delivered to mice using the ppTG-1 peptide, but the liver was not examined [Rittner et al. 2002]. When fluorescently labelled siRNA was delivered with the MPG-8 peptide, it was observed in the majority of organs with high levels in the lungs and liver [Crombez et al. 2009]. No morbidity or mortality of animals was observed following treatment in the experiments described in this work, although this has not always been the case with peptide based gene delivery agents (Rittner et al. (2002) reported the death of several mice when delivering the plasmid systemically with the ppTG1 peptide.
(174) In addition, RALA does not appear to cause a significant immune response upon repeated administration beyond the inflammation associated with tissue damage caused by the needle at the site of injection. There is also no neutralization of RALA following repeated administration. Furthermore, RALA appears to shield naked DNA from generating an adaptive immune response and does not cause an antibody response on its own. This is an encouraging result given that peptides are often used as vaccines because they share homology with viral and tumour proteins and produce a high antigenic response [Yang et al. 2009, Rodriguez and Grubman 2009]. As such, it might be expected that RALA, a peptide that is analogous to viral fusion proteins, might likewise be highly immunogenic. It appears, that as RALA uses a simple highly repetitive, artificially designed sequence that is not common in nature, its immunogenicity is low.
(175) Part of the effectiveness of RALA as a transfection agent is probably related to its ability to protect DNA or siRNA from a hostile environments. The complexation of RALA to plasmid DNA forms nanoparticles that protect DNA from, freeze-drying and degradation in serum. While the ability to protect the cargo from degradation by serum has a bearing on transfection efficacy, the ability to act as a lyoprotectant has implications for further formulation related issues that surround transfection agents. The logistics behind supplying gene medicine to clinics are complicated by the lack of stability of most prospective vectors. Since viral vectors are notoriously difficult to store and non-viral vectors usually require lyoprotectants, which alter the final formulation, before they can be successfully freeze-dried, it is promising to see that RALA/pEGFP-N1 nanoparticles retain activity following reconstitution after lyophylisation.
(176) RALA has also been shown to successfully condense and form nanoparticles with a range of bisphosphonates, siRNA and is an excellent tool for local delivery. It has also been used for the systemic delivery of the iNOS therapeutic to metastatic deposits of cancer with an excellent response. This indicates a wide range of applications for this peptide delivery system.
Example 3: Alternative Peptide Sequences
(177) The following peptide sequences based on RALA (WEARLARALARALARHLARALARALRACEA) were also prepared using conventional commercial techniques as expanded on in Example 1.
(178) TABLE-US-00008 TABLE 3 Key Characteristics RALA (WEARLARALARALARHLARALARALRACEA) derivative Peptides in ZR-75-1 breast cancer cells determined in accordance with the protocols of Example 2. Characteristics Transfection Length Efficiency Peptide SEQ ID Hydrophilic:Hydrophobic Best Size Charge in ZR-75-1 N:P10 No. +/− (nm) (mV) Cells 1. Original 1 30 mer 70 25 30% RALA 30:67:1 8:2 2. Peptide 2 2 29 mer 76 22 55 (H Removed) 31:70 7:2 3. Peptide 3 4 30 mer 51 24 41 (H Replaced 33:67 with E) 7:3 4. Peptide 4 5 29 mer 37 12 50 (H Removed 33:67 and Replaced 8:2 W replaced with R) 5. Peptide 5 6 29 mer 53 13 46 (H Removed 37:63 and W replaced 9:2 with R and C replaced with R) 6. Peptide 6 7 30 mer 308 6 43 (H Replaced 40:60 with E and W 9:3 replaced with R and C replaced with R)
Results
(179) The results in terms of transfection efficiency in ZR-75-1 cells are shown in Table 3. Peptides 1-5 successfully condensed the DNA into nanoparticles less than 100 nm. The exception being peptide 6, where the smallest nanoparticle measured was 308 nm. It can also be deduced that the highest transfection efficiency was with peptide 2 at 55% and as the hydrophilic ratios increase up to 40% the surface charge of the nanoparticle decreases. Furthermore the addition of glutamic residues reduces transfection efficiency as evidenced by peptide 3 and peptide 6. Nevertheless all sequences have potential as delivery vehicles for nucleic acids and hydrophilic compounds.
(180) A 22mer WEARLARALARALARHLRACEA was also tested but was unable to condense DNA into nanoparticles and transfect cells and was therefor deemed unsuccessful.
REFERENCES
(181) Rittner K, Benavente A, Bompard-Sorlet A, Heitz F, Divita G, Brasseur R, Jacobs E. New basic membrane-destabilizing peptides for plasmid-based gene delivery in vitro and in vivo. Mol Ther. 2002 February; 5(2):104-14. De Smedt S C, Demeester J, Hennink W E. Cationic polymer based gene delivery systems. Pharm Res. 2000 February; 17(2):113-26. Bagwe R P, Hilliard L R, Tan W. Surface modification of silica nanoparticles to reduce aggregation and nonspecific binding. Langmuir. 2006 Apr. 25; 22(9):4357-62. Murray K D, Etheridge C J, Shah S I, Matthews D A, Russell W, Gurling H M, Miller A D. Enhanced cationic liposome-mediated transfection using the DNA-binding peptide mu (mu) from the adenovirus core. Gene Ther. 2001 March; 8(6):453-60. Malim M H, Hauber J, Le S Y, Maizel J V, Cullen B R. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989 Mar. 16; 338(6212):254-7. Crombez L, Morris M C, Dufort S, Aldrian-Herrada G, Nguyen Q, Mc Master G, Coll J L, Heitz F, Divita G. Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth. Nucleic Acids Res. 2009 August; 37(14):4559-69. Yang T, Wang H N, Wang X, Tang J N, Lu D, Zhang Y F, Guo Z C, Li Y L, Gao R, Kang R M. The protective immune response against infectious bronchitis virus induced by multi-epitope based peptide vaccines. Biosci Biotechnol Biochem. 2009 July; 73(7):1500-4. Rodriguez L L, Grubman M J. Foot and mouth disease virus vaccines. Vaccine. 2009 Nov. 5; 27 Suppl 4:D90-4.