Transfection and Transduction System

Abstract

The invention relates to the fabrication and use of silica organic nanoparticles as delivery vehicles for vims and virus-like species to the body. The nanoparticles typically have a hollow core and a surface morphology that allows effective adhesion of species to the surface for delivery to the body. In particular, the invention is particularly useful for performing transfection and transduction.

Claims

1. A composition comprising: an inorganic mesoporous nanoparticle comprising silica; and one or more delivery components; wherein the nanoparticle comprises projections thereon; wherein the nanoparticle has a diameter in the range 50 nm to 3000 nm; wherein the inorganic nanoparticle is at least partially coated with a transfection agent; and wherein the one or more delivery components comprises a viral vector.

2. The composition according to claim 1, wherein the viral vector is an adenoviral vector, an ademo-associated viral vector, or a retroviral vector.

3. The composition according to claim 1, wherein the nanoparticle comprises: a shell comprising silica; a hollow core with a volume defined by the inner surface of the shell; and a plurality of projections comprising silica disposed on the exterior of the shell.

4. (canceled)

5. The composition according to claim 1, wherein the nanoparticle is hollow.

6. The composition according to claim 1, wherein the nanoparticle is rambutan-like or morningstar-like.

7. The composition according to claim 1, wherein the projections comprise fingers or spikes.

8. The composition according to claim 1, wherein the transfection agent is a cationic polymer.

9. The composition according to claim 8, wherein the transfection agent is a polyalkylimine, chitosan, polylysine, DEAE-dextran, polybrene, or polyamidoamine (PAMAM) dendrimer.

10. The composition according to claim 1, wherein the nanoparticle is at least partially coated with the one or more delivery components.

11. The composition according to claim 1, wherein the nanoparticle comprises at least two delivery components.

12-14. (canceled)

15. The composition according to claim 1, further comprising a cell.

16. The composition according to claim 15, wherein the cell is a CAR-T cell or a TCR-T cell.

17-20. (canceled)

21. A method of treating a disease in a patient, comprising administering the composition according to claim 1 to the patient.

22. A method of manufacturing the composition according to claim 1, the method comprising the step of: i) mixing an inorganic mesoporous nanoparticle comprising silica and having projections thereon with one or more delivery components; wherein the diameter of the nanoparticles are in range of from 50 nm to 3000 nm; and wherein the one or more delivery components comprises a viral vector.

23. A method of transfecting or transducing a cell comprising the steps of: (i) providing a composition according to claim 1; and (ii) incubating the composition with a cell.

24. (canceled)

25. (canceled)

26. The composition according to claim 2, wherein the viral vector is an adenoviral vector or a lentiviral vector.

27. The composition according to claim 3, wherein the projections are integral with the shell.

28. The composition according to claim 3, wherein the projections extend radially outwards from the shell.

29. The composition according to claim 1, wherein the projections have a length of 5 nm to 1000 nm, from 10 nm to 200 nm, or from 50 nm to 150 nm.

30. The composition according to claim 9, wherein the transfection agent is a polyalkylimine or a polyethylimine (PEI).

31. The method of claim 21, wherein the disease is a genetic disorder, cancer, infection, or autoimmune disease.

Description

DESCRIPTION OF FIGURES

[0077] The invention will now be described in detail by way of example only with reference to the figures which are as follows:

[0078] FIG. 1. Cell viability after 4 h treatment with Nuvec®®. Viability of cells treated with different concentrations of Nuvec®® (Negative Control (NC), 1 μg, 10 μg, 40 μg, 60 μg, 80 μg) for 4 h. After 4 h, the medium was changed. Cell survival and recovery was recorded for 72 h post-medium change, at the time points shown.

[0079] FIG. 2. Cell viability after 4 h treatment with Nuvec®®. (A, B) Cell survival over time (every 24 h) of HEK293T cells treated with a range of concentrations of Nuvec® for 4 h (n=1). See Table 3 below.

[0080] FIG. 3. Cell viability after 24 h treatment with Nuvec®. Viability of cells treated with different concentrations of Nuvec® (Negative Control (NC), 1 μg, 10 μg, 40 μg, 60 μg, 80 μg) for 24 h. After 24 h, the medium was changed. Cell survival and recovery was recorded for 72 h post-medium change, at the time points shown.

[0081] FIG. 4. Cell viability after 24 h treatment with Nuvec®. (A, B) Cell survival over time of HEK293T cells treated with a range of concentrations of Nuvec® for 24 h. See Table 4 below.

[0082] FIG. 5. Cell viability after 48 h treatment with Nuvec®. Viability of cells treated with different concentrations of Nuvec® (Negative Control (NC), 1 μg, 10 μg, 40 μg, 60 μg, 80 μg) for 48 h. After 48 h, the medium was changed. Cell survival and recovery was recorded for 72 h post-medium change, at the time points shown.

[0083] FIG. 6. Cell viability after 48 h treatment with Nuvec®. (A, B) Cell survival over time of HEK293T cells treated with a range of concentrations of Nuvec® for 48 h.

[0084] FIG. 7. Cell viability and GFP expression after 4 h, 24 h and 48 h transfection with 1 μg Nuvec® and 5 μg GFP vector only. Analysis at 72 h post-transfection.

[0085] FIG. 8. Cell viability and GFP expression after 4 h, 24 h and 48 h transfection with 10 μg Nuvec® and 5 μg GFP vector only. Analysis at 72 h post-transfection.

[0086] FIG. 9. Cell viability and GFP expression after 4 h, 24 h and 48 h transfection with 40 μg Nuvec® and 5 μg GFP vector only. Analysis at 72 h post-transfection.

[0087] FIG. 10. Cell viability and GFP expression after 4 h, 24 h and 48 h transfection with 60 μg Nuvec® and 5 μg GFP vector only. Analysis at 72 h post-transfection.

[0088] FIG. 11. Cell viability and GFP expression after 4 h, 24 h and 48 h transfection with 80 μg Nuvec® and 5 μg GFP vector only. Analysis at 72 h post-transfection.

[0089] FIG. 12. Viruses produced and titrated against indicator cells to use later to test coupling with Nuvec®. High titre LV was produced using traditional production methods and titrated using various dilutions of LV. Analysis via flow cytometry determined percentage expression of GFP at various dilutions which was used to calculate a titre of 1.18×10.sup.9TU/ml. LV was produced for future use with Nuvec® for transduction assays.

[0090] FIG. 13. HEK293T indicator cells were transfected using NV00100028, NV00100026-28, NV0010032 and NV0010033 with 5 μg of plasmid DNA; GFP, GAG-POL and VSV-G at a ratio of 4:3:1, for either 4 h or 24 h. Viral supernatant was harvested every 24 h for 72 h. Photos show transfected cells 72 h post-transfection.

[0091] FIG. 14. HEK293T cells transduced with viral supernatant collected of cells transfected using Nuvec® at different concentrations and transfection times. Percentage of GFP positive cells were analysed using flow cytometry 72 h post transduction and are shown below each image. Initially, it was unknown how much lentivirus Nuvec® could actually generate and 500p1 and 200p1 of viral supernatant was used to transduce HEK293T cells. For a more true representation of viral titres the calculation for lentivirus titres use percentages between 1-30% GFP positive. As shown above even at the lowest dilution used (200 p1) cells were still more than 30% GFP+ so repeats using smaller volumes was carried out.

[0092] FIG. 15. Repeat titrations were carried out using smaller volumes of viral supernatant collected of cells transfected with different concentrations of Nuvec®. HEK293T cells were then transduced with 100p1 and 10p1 of viral supernatant and percentage of positive green cells was determined using flow cytometry 72 h post-transduction.

[0093] FIG. 16. Analysis of lentivirus transduction efficiency on indicator cells. Complexation of lentivirus (MOI 20), with various concentrations of NV00100028. Complexation occurred for various incubation times before adding lentivirus to HEK293T indicator cells. N=4

[0094] FIG. 17. Analysis of lentivirus transduction efficiency on indicator cells. Complexation of lentivirus (MOI 20), with various concentrations of NV00100026-28. Complexation occurred for various incubation times before adding lentivirus to HEK293T indicator cells. N=4

[0095] FIG. 18. Analysis of lentivirus transduction efficiency on indicator cells. Complexation of lentivirus (MOI 20), with various concentrations of NV0010032. Complexation occurred for various incubation times before adding lentivirus to HEK293T indicator cells. N=4

[0096] FIG. 19. Analysis of lentivirus transduction efficiency on indicator cells. Complexation of lentivirus (MOI 20), with various concentrations of NV0010033. Complexation occurred for various incubation times before adding lentivirus to HEK293T indicator cells. N=4

[0097] FIG. 20. Transduction efficiency of lentivirus (MOI 20) on indicator cells. Lentivirus was complexed with various batches of Nuvec®, at various concentrations for 10 min before addition to HEK293T indicator cells. GFP expression was measured via flow cytometry. N=4

[0098] FIG. 21. Transduction efficiency of lentivirus (MOI 20) on indicator cells. Lentivirus was complexed with various batches of Nuvec®, at various concentrations for 20 min before addition to HEK293T indicator cells. GFP expression was measured via flow cytometry. N=4

[0099] FIG. 22. Transduction efficiency of lentivirus (MOI 20) on indicator cells. Lentivirus was complexed with various batches of Nuvec®, at various concentrations for 30 min before addition to HEK293T indicator cells. GFP expression was measured via flow cytometry. N=4

[0100] FIG. 23. Average cell viability after transduction of lentivirus (MOI 20) with various concentrations of Nuvec® and various incubation times. N=2.

[0101] FIG. 24. Virus transduction of indicator cells+/−polybrene. Transduced cells using lentivirus at various limiting dilutions, with or without 5 μg/ml of polybrene. See Table 7.

[0102] FIG. 25 shows SEM images of the nanoparticles.

EXAMPLES

Example 1—Synthesis of Ram-SNPs

[0103] Resorcinol (0.4 g) and formaldehyde (37 wt %, 0.56 mL) were added to the solution composed of ammonia aqueous solution (28 wt %, 12 mL), deionized water (40 mL) and ethanol (280 mL). The mixture was stirred for 8 h at room temperature (−25° C.), then 2.4 mL of tetraethylorthosilicate (TEOS) was added to the solution and stirred for 8 minutes before the second addition of resorcinol (1.6 g) and formaldehyde (37 wt %, 2.24 mL). The mixture was stirred for 2 h at room temperature, and then collected by centrifugation at 4700 rpm (3877 rcf) for 5 min, washed with ethanol and dried at 50° C. overnight. Finally, Ram-SNPs were collected by calcination at 550° C. for 5 h in air.

Example 2—Synthesis of PEI Modified RNPs

[0104] 0.03 gram of Ram-SNPs fabricated above were dispersed into 10 mL water under ultrasonication (ensure the no obvious bulk particles sediment in the solution). 0.213 mL of 3-(Trihydroxysilyl)propyl methylphosphonate (HTPMP, 50 wt % in water) was added into another 10 mL of water, and then the 10 mL of nanoparticle solution mixed with HTPMP solution, and t stirred at 40° C. for 2 h. The phosphonate modified silica nanoparticles were collected by centrifugation at 12000 rpm (17,420 rcf) for 5 min, and washed by water once. These nanoparticles were then directly re-dispersed into 5 mL carbonate-bicarbonate buffer solution (pH=9.6) by ultrasonication.

[0105] Polyethylenimine (PEI, branched, mean MW of 10 k, Alfar Aesar) was dissolved into 10 mL of carbonate-bicarbonate buffer under ultrasonication. Then the particle solution and PEI solutions were mixed together and stirred at room temperature for 4 h. The PEI loaded nanoparticles were collected by centrifugation at 12000 rpm for 5 min, and then washed by water once. The nanoparticles were then resuspended into 3 mL of water, and frozen under liquid nitrogen for 30 min, followed by drying at freeze-dryer for 2 days. The thoroughly dried particles were stored in the fridge in a desiccator and are hereinafter referred to as “Nuvec®”.

Example 3—Cell Viability

[0106] 6×10.sup.5 HEK293T cells were seeded in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin Streptomycin (hereinafter termed “complete medium”) and incubated at 37° C., 5% CO.sub.2 overnight.

[0107] Cells were treated with various concentrations of Nuvec ranging from 1 μg to 80 μg/0.5 ml. Treated cells were incubated at 37° C., 5% CO.sub.2 for either 4 h, 24 h or 48 h before replacing with fresh complete medium. Cell viability was measured every 24 h post medium change for 72 h via a trypan blue exclusion assay. A Countess™ automated cell counter was used according to manufacturer's instructions.

[0108] Cell viability analysis following a 4 h incubation with Nuvec is provided in FIGS. 1 and 2. Cell viability analysis following a 24 h incubation with Nuvec is provided in FIGS. 3 and 4. Cell viability analysis following a 48 h treatment with Nuvec is provided in FIGS. 5 and 6.

Example 4— Single Plasmid Transfection Using Nuvec

[0109] The transfection efficacy of Nuvec was evaluated in HEK293T cells by assessing delivery efficiency of a plasmid DNA encoding green fluorescent protein (pDNA-EGFP).

[0110] Fresh HEK293T cells were seeded into 12 well plates at a density of 1.2×10.sup.5 cells per well in complete medium and incubated at 37° C., 5% CO.sub.2 overnight to achieve 70-90% confluency.

[0111] Various amounts of Nuvec, ranging from 1-80 μg per well, were treated to minimise bioburden by resuspending the particles in at least 5 volumes of 70% ethanol, mixing, centrifuging briefly to collect the particles, then removing the clear supernatant. The suspension of Nuvec in PBS was then prepared by adding 50 μl sterile PBS to each well and sonicating using a bath sonicator with an output of at least 120 W until a homogeneous suspension was achieved (up to 30 min). Any fast-settling clumps during the sonication procedure were dispersed by pipetting.

[0112] The various concentrations of Nuvec in PBS, ranging from 1 μg to 80 μg/0.5 ml, were complexed with a total of 1 μg of plasmid DNA for 30 min at room temperature. After complexation, the pDNA loaded Nuvec was then suspended into 1 ml of complete medium and used to replace the cell culture medium. Cell were incubated at 37° C., 5% CO.sub.2 for 4 h, 24 h or 48 h, before replacing with fresh complete medium. Cells were analysed at 72 h post medium change by flow cytometry and confocal microscopy to determine the green fluorescent protein expression in cells. Experiments were performed in triplicate for each group. Results are provided in FIGS. 7-11.

Example 5—Production of Lentivirus

[0113] 1.5×10.sup.7 fresh HEK293T cells were seeded in a T175 flask in complete medium and incubated at 37° C., 5% CO.sub.2 overnight to achieve confluency.

[0114] A total of 5 μg plasmid DNA (eGFP, pCMVR8.74 and pMD2.G) at a ratio of 4:3:1 was complexed with polybrene for 20 min in serum free medium (Opti-MEM). After complexation, the pDNA loaded transfection reagent was suspended in 1 ml complete medium and used to replace the cell culture medium. Cells were incubated at 37° C., 5% CO.sub.2 for 24 h before replacing with fresh complete medium. Supernatant was harvested every 24 h for 72 h post replacement. Conditioned medium was filtered through 0.45 μM filters to remove cell debris and stored at 4° C. for future use.

[0115] Conditioned medium was concentrated via ultracentrifugation at 23,000 rpm at 4° C. The supernatant was discarded and the pellet air dried for 10 min. The pellet was resuspended in 200 μl serum free medium and incubated on ice for 1 h. The resuspended viral pellet was aliquoted and stored at −80° C. for future use.

Example 6— Lentivirus Titration

[0116] Fresh HEK293T cells were seeded into 12 well plates at a density of 2×10.sup.5 cells/well in complete medium and incubated at 37° C., 5% CO.sub.2 overnight.

[0117] Serial dilutions of virus were prepared and incubated in complete medium with 5 μg/ml polybrene for 20 min at room temperature. The virus polybrene mixture was then suspended into 1 ml complete medium and used to replace the cell culture medium. One well of cells was left as untreated and one well of cells were counted to determine the number of cells present for infection.

[0118] Cells were incubated at 37° C., 5% CO.sub.2 for 24 h before replacing with fresh complete medium. Cells were analysed at 48 h post medium replacement by flow cytometry analysis of GFP expression. Only samples expressing 1-30% GFP expression were analysed as accurate representations of viral titre. Virus titre was calculated at each dilution point, as shown below, and averaged for overall average titre.

Titre(TU/ml)=((Cell count*(Percentage GFP expression/100))/Volume)*DF

[0119] TU/ml is transduction units per ml. DF is the dilution factor. FIG. 12 shows images of GFP expression at limiting dilutions. A titre of 1.18×10.sup.9TU/ml was calculated.

Example 7— Triple Plasmid Transfection of Cells

[0120] Fresh HEK293T cells were seeded into 6 well plates at a density of 6×10.sup.5 cells per well in complete medium and incubated at 37° C., 5% CO.sub.2 overnight to achieve confluency.

[0121] Various concentrations of Nuvec, ranging from 1 μg to 80 μg/0.5 ml, were complexed with a total of 5 μg of plasmid DNA (eGFP, pCMVR8.74 and pMD2.G) at a ratio of 4:3:1 for 30 min in serum free medium. Different batches of Nuvec were also tested. After complexation, the pDNA loaded Nuvec was suspended in 1 ml complete medium and used to replace the cell culture medium. Cells were incubated at 37° C., 5% CO.sub.2 for either 4 h or 24 h before replacing with fresh complete medium. Supernatant was harvested every 24 h for 72 h post medium replacement. Cells were analysed at 72 h post-transfection for GFP expression using fluorescence microscopy. Conditioned medium was centrifuged at 1500 rpm for 5 min with the supernatant collected and stored at 4° C. for future use. FIG. 13 shows transfected cells 72 h post-transfection.

[0122] Transfection experiments were repeated a total of three times for NV00100028, NV00100026-28, NV0010032 and NV0010033 as well as 4 hour and 24 hour transfections. The titres generated (TU/ml) were calculated and are shown in the table below.

TABLE-US-00001 TABLE 1 Transfection Experiments (all values are “×10.sup.4”). 1 μg 10 μg 40 μg 60 μg 80 μg N 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 NV00100028 0 0 0 0 0 0 3.02 4.21 3.25 61.6 8.48 4.65 58.1 131 31.1 4 hours NV00100028 0 0 0 0 0 0 06.7 3.97 5.83 149 13.3 8.72 104 221 32.4 24 hours NV00100026-28 0 0 0 0 0 0 5.36 3.65 0 130 4.51 0 64.0 0 0 4 hours NV00100026-28 0 0 0 0 0 0 2.97 2.81 0 213 3.38 0 124 0 0 24 hours NV0010032 0 0 0 0 0 0 22.3 0 0 56.1 2.92 2.75 68.2 12.6 N/A 4 hours NV0010032 0 0 0 0 0 0 17.9 0 0 52.5 4.70 4.35 52.2 16.3 6.88 24 hours NV0010033 0 0 0 0 0 0 3.02 0 0 0 0 0 0 0 0 4 hours NV0010033 0 0 0 0 0 0 0.67 0 0 4.30 4.70 N/A 15.1 16.3 0 24 hours

Example 8—Transduction of Cells with Conditioned Medium

[0123] Fresh HEK293T cells were seeded into 12 well plates at a density of 2×10.sup.5 cells/well and incubated at 37° C., 5% CO.sub.2 overnight to achieve confluency.

[0124] Cells were transduced with 500p1 or 200p1 conditioned medium collected from cells transfected in Example 7. Cells were incubated at 37° C., 5% CO.sub.2 for 24 h before replacing with fresh complete medium. Cells were analysed at 72 h post-transduction for GFP expression using fluorescence microscopy and flow cytometry. The results are provided in FIG. 14.

[0125] Fresh HEK293T cells were transduced with 100p1 or 10p1 conditioned medium collected from cells transfected in Example 7. Cells were incubated at 37° C., 5% CO.sub.2 for 24 h before replacing with fresh complete medium. Cells were analysed at 72 h post transduction for GFP expression using fluorescence microscopy and flow cytometry. The results are provided in FIG. 15.

Example 9—Transduction with Lentivirus

[0126] Fresh HEK293T cells were seeded into 12 well plates at a density of 2×10.sup.5 cells/well in complete medium and incubated at 37° C., 5% CO.sub.2 overnight to achieve confluency.

[0127] Various concentrations of Nuvec, ranging from 1 μg to 80 μg/0.5 ml, were complexed with lentivirus carrying a GFP transgene (MOI 20) for 10 min, 20 min or 30 min at room temperature. The lentivirus was made according to Example 5. Different batches of Nuvec® were tested.

[0128] After complexation, the lentivirus and Nuvec mixture was suspended in 1 ml complete medium and used to replace the cell culture medium. Cells were incubated at 37° C., 5% CO.sub.2 for 24 h before replacing with fresh complete medium. Cells were analysed at 72 h post-transduction for GFP expression using fluorescence microscopy and flow cytometry. FIGS. 16-22 show GFP expression of transduced cells using different batches of Nuvec.

[0129] Viability of transduced cells was analysed using a trypan blue exclusion assay. The results are provided in FIG. 23.

Example 10—Comparison with Standard Transduction Protocol

[0130] Fresh HEK293T cells were seeded into 12 well plates at a density of 2×10.sup.5 cells/well in complete medium and incubated at 37° C., 5% CO.sub.2 overnight to achieve confluency.

[0131] Various limiting dilutions of lentivirus carrying a GFP transgene (MOI 20) was complexed with 5 μg/ml polybrene for 20 mins at room temperature. Lentivirus incubated in the absence of polybrene was used as a control.

[0132] After complexation, the lentivirus and polybrene mixture was suspended in 1 ml complete medium and used to replace the cell culture medium. Cells were incubated at 37° C., 5% CO.sub.2 for 24 h before replacing with fresh complete medium. Cells were analysed at 72 h post-transduction for GFP expression using fluorescence microscopy and flow cytometry. FIG. 24 shows GFP expression of transduced cells.

[0133] A summary of the transduction efficiency is provided in the table below.

TABLE-US-00002 TABLE 2 Transduction Efficiency Information. Nuvec ® concentration/μg Batch 1 μg 10 μg 40 μg 60 μg 80 μg 10 minutes NV00100028 3.14 ± 0.21 3.02 ± 0.15  8.36 ± 2.31 17.92 ± 3.90 24.76 ± 2.99 NV00100026-28 1.51 ± 0.17 3.20 ± 0.26 19.81 ± 3.61 27.71 ± 2.04 40.33 ± 2.46 NV0010032 3.65 ± 1.32 4.87 ± 0.88 15.35 ± 4.30 29.38 ± 3.80 36.03 ± 4.37 NV0010033 1.43 ± 0.33 5.22 ± 0.51  7.30 ± 4.24 17.11 ± 5.04 40.09 ± 2.01 20 minutes NV00100028 3.32 ± 0.96 2.98 ± 0.58  8.77 ± 1.40 17.55 ± 2.33 24.81 ± 3.22 NV00100026-28 1.90 ± 0.64 2.88 ± 0.73 13.86 ± 6.30  24.44 ± 10.83  31.83 ± 11.04 NV0010032 5.47 ± 1.15 5.97 ± 1.52 15.99 ± 5.06 26.97 ± 4.56 31.20 ± 3.24 NV0010033 1.64 ± 0.24 4.08 ± 1.21  5.73 ± 2.51  9.80 ± 4.93  18.76 ± 11.06 30 minutes NV00100028 2.11 ± 0.23 2.91 ± 0.42 10.02 ± 3.13 15.39 ± 3.46 20.77 ± 3.78 NV00100026-28 1.56 ± 0.14 3.51 ± 0.52 17.34 ± 2.42 35.03 ± 5.57 39.97 ± 6.05 NV0010032 1.17 ± 0.14 2.55 ± 0.25  7.99 ± 2.27 15.20 ± 5.63 24.30 ± 7.02 NV0010033 2.81 ± 0.23 7.12 ± 0.88 10.47 ± 2.66 21.42 ± 2.80 52.11 ± 3.55 +Polybrene 2.61 ± 0.46 −Polybrene  1.40 ± 0.40

TABLE-US-00003 TABLE 3 Viability data of cells treated in accordance with FIG. 2. Viability 4 Viability 24 Viability 48 Viability 72 hours post hours post hours post hours post Concentration treatment treatment treatment treatment 0 95% 98% 97% 98%  1 μg 93% 96% 97% 98% 10 μg 89% 96% 97% 97% 40 μg 77% 93% 95% 98% 60 μg 71% 88% 92% 91% 80 μg 62% 83% 80% 91%

TABLE-US-00004 TABLE 4 Viability data of cells treated in accordance with FIG. 4. Viability 24 Viability 48 Viability 72 Viability 96 hours post hours post hours post hours post Concentration treatment treatment treatment treatment 0 93% 97% 98% 96%  1 μg 95% 96% 96% 94% 10 μg 92% 98% 96% 93% 40 μg 84% 94% 96% 84% 60 μg 70% 90% 77% 87% 80 μg 60% 81% 77% 80%

TABLE-US-00005 TABLE 5 Viability data of cells treated in accordance with FIG. 6. Viability 48 Viability 72 Viability 96 Viability 120 hours post hours post hours post hours post Concentration treatment treatment treatment treatment 0 97% 93% 93% 98%  1 μg 96% 93% 95% 97% 10 μg 94% 92% 92% 96% 40 μg 92% 90% 86% 97% 60 μg 82% 76% 75% 95% 80 μg 78% 72% 64% 60%

TABLE-US-00006 TABLE 6 Dilutions as shown in FIG. 12. Dilution % GFP Titre/ml × 10.sup.8 1 84.12 4.33 1 83.38 4.29 −1 27.57 14.2 −1 25.47 13.1 −2 3.36 17.3 −2 3.4 17.5 −3 0.81 41.7 −3 0.91 46.9 −4 1.03 530 −4 0.59 304

TABLE-US-00007 TABLE 7 Dilutions as shown in FIG. 24. 1 × 1 × 1 × 1 × Dilution 1 μl 10.sup.−1 μl 10.sup.−2 μl 10.sup.−3 μl 10.sup.−4 μl With Pb 66 ± 0.2 20 ± 1   3 ± 0.1 3 ± 0.1    1 ± 0.3 Without Pb 43 ± 0.8 6 ± 0.4 1 ± 0.4 2 ± 0.26 0.7 ± 0.1