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The use of a scanning proton microprobe in AIDS research
Nuclear Instruments and Methods in Physics Research B77 (1993) 282-286 North-Holland
NOM6
Beam Interactions with Materials&Atoms
The use of a scanning proton microprobe in AIDS research M. Cholewa ’ and G.J.F. Legge Micro Analytical Research Centre (MARC), School of Physics, The University of Melbourne, Parkuille, EC. 3052, Australia
H. Weigold and G. Holan Division of Chemicals and Polymers, CSIRO, Private Bag 10, Clayton, Australia
C. Birch virology Department, Fairfield Hospital, Fairfield, Australia
A series of organometallic and inorganic drugs has been synthesized at the CSIRO Division of Chemicals and Polymers. The drugs, which are all polyanions of various size, shape and charge are being tested for their activity for the HIV virus in a continuous human T-lymphocyte line (MT21 and in peripheral blood lymphocytes (PBLs). Determinations of drug activity have been carried out at the Fairfield Hospital’s Virology Department. It is important for the drug synthesis programme to develop an understanding of the relationship between polyanion properties and antiviral activity. For this it is essential to establish: (a) whether polyanions enter HIV infected cells, (b) their distribution within these cells, (c) whether this distribution is the same for all polyanions, (d) whether the drugs remain intact (do not dissociate) on entering the cell, (e) the differences between active and inactive drugs of similar structure. Answers to these questions and to others will facilitate the synthesis programme.
1. Introduction
This paper describes work in which the distinctive features of an analytical technique proton induced X-ray emission (PIXE) [l], the versatility of a microscopical instrument (the ion microprobe) [2] and the power of a data handling method (TQSA) [31 combine to handle an otherwise intractable problem in medicine and pharmacology. There is an urgent need to develop satisfactory inhibitors of the human immunodeficiency virus (HIV), causative agent of the acquired immunodeficiency syndrome (AIDS). All currently used drugs have serious side effects. Any antiviral drug is likely to be toxic also to normal cells. The aim in any drug development programme that is based on attacking the virus directly must be to maximise the ratio of toxicity to virus versus toxicity to normal cell and to optimise the delivery of the drug to the site of the virus. A team of chemists at the CSIRO Division of Chemicals and Polymers in Melbourne has developed a line of drugs which are toxic to HIV [4]. These drugs are tested by scientists at the Fairfield Hospital Virology Department for their toxicity to the virus and to normal blood cells. This work alone is very extensive and gives no 1 Permanent
address: Institute of Nuclear Physics, Cracow,
Poland. 0168-583X/93/$06.00
indication as to why one drug may be more effective than another or more toxic to normal cells, while another drug which is closely related chemically may show little effect. What is lacking is any evidence on the relative penetration of the drug into the cells (i.e. are the differences in activity between similar drugs due in part to the variations in their transport efficiency through the cell membranes?) and on their composition within the cell (i.e. has the structure of the applied drug been retained within the cell?). This information would be most valuable for the design of drugs of optimal activity. The drugs are designed to inhibit the replication of HIV in T-lymphocytes. They are tested for this activity and also for their toxicity to healthy cells in a continuous human T-lymhocyte line (MT2 cells) and in peripheral blood lymphocytes (PBLs) from healthy donors. A characteristic of these drugs essential to this investigation is that, apart from the usual light elements found in most drugs, they contain also heavy metal atoms which are not normally seen in cells. These can act as tags to identify the drugs. Unfortunately the drugs are toxic to normal cells and concentrations must therefore be kept below 5 to 200 yg per ml of culture medium, the exact level depending on the drug. The heavy metal concentration within the cell is then well below that measurable with an electron microprobe, but is ideally
0 1993 - Elsevier Science Publishers B.V. All rights reserved
283
M. Cholewa et al. / The use of a scanning proton microprobe in AIDS research
suited to a proton microprobe, because of the extremely low bremsstrahlung level in a PIXE spectrum at the high energy end - that of the so-called heavy metals. What is more, where there are two identifiable tags in the spectrum (from two heavy metals in the drug), a comparison of their ratio within the cell and in the pure drug will indicate whether the drug remains intact within the cell. Finally it is possible to map the distribution of the drug within the cell and its nucleus. This requires a spatial resolution of about 1 micron. The emphasis in this paper is on the techniques and practical details of the microprobe operations involved.
600 E 500 g 400 300
0 2.0
2. Experiment Prior to measurements with the blood cells, PIXE spectra were measured for the pure drugs to be tested. This was partly in order to compare the measured elemental contents with those expected from the design formulae of the synhesised drugs. But the major reason was to establish a quantitative fingerprint for the drug that could be compared with the spectra taken from the cells. In order to avoid unknown energy loss and X-ray absorption in an unknown sample thickness, it was necessary to use either thick or very thin samples of the drugs. Whilst the thick sample effects were in theory calculable, it was decided that quantitative accuracy would be more readily achieved with very thin samples, for which no correction to the spectrum was required. These measurements were more difficult than anticipated, because the drugs were to hard and brittle to section with a microtome and, when solutions of the drugs were allowed to evaporate on nylon foils, the microscopic crystals that formed were not thin enough to totally avoid absorption effects, as evidenced by the nonreproducible spectra obtained. Success was finally achieved by evaporating very weak solutions of
4.0
10.0
6.0 ENERGY (k::)
12.0
Fig. 2. A typical elemental spectrum from a single PBL exposed to 50 yg/ml of GH1532 (KdCo,(H,O>,(PW,O,),] .H,O) drug. The detected concentration of tungsten ( - 10 ppm) in the cell is much above the detection limit of the SPMP, but the level of the drug in the culture medium was a factor of 4 lower than the toxic level.
the drugs in alcohol. Fig. 1 shows a spectrum from one of the drugs, GH1532, whose formula is K,,[Co, (H,O),O'W,O,,~,l . H,O. The lymphocytes were incubated with growth medium containing the drug at various subtoxic levels and this was then removed by resuspending in a buffer of ammonium acetate. Droplets of the cell suspension were placed on nylon foils (mounted on aluminium frames), immediately snap frozen in isopentane cooled by liquid nitrogen and then transferred at liquid nitrogen temperature to a high vacuum vessel in which they were freeze dried. We had previously shown this buffer to be well suited to such work [5], because the ammonium salt evaporates with no trace during the freeze drying process. However, the lymphocytes could not tolerate long exposure to this buffer; so extracellular traces of the drug were removed by repeated centrifu-
2000
,
,
,
,
,
,
,
,
8
100 E
60 60
0 2.0
4.0
6.0 6.0 ENERGY (keV)
Fig. 1. A typical elemental
spectrum
12.0
10.0
from
a GH1532
(K,D[CO,(H~O),(PW,O,~)~I.H~O) drug.
Fig. 3. A typical energy spectrum fr0rn.a single control PBL cell. VI. BIOMEDICAL
284
M. Cholewa et al. / The use of a
scanningproton microprobe in AIDS research
gation in pure growth medium, with a final resuspension in the ammonium acetate (to remove growth medium) requiring exposure for a period of only about 10 min. For a control sample and for samples exposed to a drug at various subtoxic levels, individual lymphocytes were scanned with the microprobe beam of 3 MeV protons. As the approximately 10 micron diameter cells freeze dry on the 1 micron thick nylon foil to discs of approximately 12 micron diameter and 1 micron thickness, there was no need of thick sample corrections but there was need of long exposure to the beam and
Fig. 4. Distribution
of phosphorus,
cobalt
and tungsten
hence good mechanical stability. This was exacerbated by the unusual beam sensitivity of these cells, which would tolerate only 30 pA of beam. A continuously moving, 1 micron diameter beam spot was scanned over the cell for approximately 2 h and all PIXE data were collected by TQSA. Spectra and all elemental maps could be observed during the data collection, so that any errors in identifying and centering healthy cells or any movements could be detected. Fig. 2 shows the spectrum extracted from a cell exposed to the drug GH1532 at a level of 50 ug/ml. The heavy metal elements, Co and W, seen in fig. 1 are again clearly
inside cells when exposed medium.
to a subtoxic
level of GH1.532 drug in the culture
M. Cholewa et al. / The use of a scanning proton microprobe in AIDS research
visible, though the light elements of the drug are swamped by those of the cell. Fig. 3 shows the clear spectrum at high energies for a control cell. If the spectra from many cells are combined, it can be shown that the drug remains intact within the cells, or that the ratio of Co to W at least does not change. The intracellular distributions of these elements can also be mapped and those of P, Co and W are shown in fig. 4. The P tends to concentrate in the cell nucleus and therefore serves to identify this organelle. Hence it is possible to extract spectra for the nucleus and the cytoplasm independently, to measure the intranuclear component of the drug. This requires the accumulation of statistics from a large nuinber of cells.
285
Knowing that these cells were extremely sensitive to irradiation, we examined them with scanning transmission ion microscopy @TIM) [6]. A 2 MeV beam of alpha particles was used in order to maximise the density sensitivity. This beam was focused to less than 200 nm and scanned over a cell. The bright field median image shown in fig. 5a was smoothed with a Gaussian of FWHM 500 nm. In order not to disturb the cell during the PIXE irradiation, the same beam was used, but with 100 pA at 1 micron resolution - a severe exposure which is close to the limit for these cells. Fig. 5b shows a second STIM image of this cell, collected after PIXE irradiation. Quantitative measurements on these two sets of data show that the cell
Fig. 5. STIM maps collected before (A) and after (B) PIXE analysis. Both STIM and PIXE analysis were performed with 2 MeV OL+beams. VI. BIOMEDICAL
286
M. Cholewa et al. / The use of a scanning
shrank in diameter by about 1 micron and in thickness by about 0.2 micron. They also show a sideways movement of about 1 micron. However, the movement and the shrinkages, associated with the loss of light matrix elements [7], would be insufficient to affect the 1 micron resolution of the PIXE data. In order to detect the heavy metal drug elements, it was necessary to use a proton beam and the beam current was also much lower; therefore, for these measurements, the effects would have been much less.
3. Conclusion The measurements reported in this paper are but the first in a series to be carried out in a programme to synthesize drugs with maximum activity against HIV and minimum toxicity to healthy cells. The techniques developed to prepare and handle the blood cells and to make the necessary microprobe measurements have been successful and the heavy metal content of these drugs have proved to be adequate for the identification and quantitative mapping of these drug distributions within their target cells. The measurements are lengthy, in order to collect significant statistics, and the X-ray
proton microprobe in AIDS research collection efficiency is soon to be increased by an order of magnitude with the installation of a new, large area Si(Li) detector.
Acknowledgements We gratefully acknowledge the support of a CSIRO/ University of Melbourne research grant and a Commonwealth AIDS Research Grant.
References [ll T.B. Johansson, R. Akselsson and S.A.E. Johansson, Nucl. Instr. and Meth. 84 (1970) 141. [2] J.A. Cookson, A.T.G. Ferguson and F.D. Pilling, J. Radianal. Chem. 12 (1972) 39. [3] G.J.F. Legge and I. Hammond, J. Microsc. 117 (1979) 201. [4] H. Weigold, G. Holan, S.M. Marcuccio, I.D. Gust and C.J. Birch, Int. Patent Appl., PCT/AU/00280, 28 June 1991. [5] G.L. Allan, J. Camakaris and G.J.F. Legge, Nucl. Instr. and Meth. B54 (1991) 175. [6] J.C. Gverley, R.C. Connolly, G.E. Sieger, J.D. MacDonald and H.W. Lefevre, Nucl. Instr. and Meth. 218 (1983) 43. [7] M. Cholewa, G. Bench, B.J. Kirby and G.J.F. Legge, Nucl. Instr. and Meth. B54 (1991) 101.
NOM6
Beam Interactions with Materials&Atoms
The use of a scanning proton microprobe in AIDS research M. Cholewa ’ and G.J.F. Legge Micro Analytical Research Centre (MARC), School of Physics, The University of Melbourne, Parkuille, EC. 3052, Australia
H. Weigold and G. Holan Division of Chemicals and Polymers, CSIRO, Private Bag 10, Clayton, Australia
C. Birch virology Department, Fairfield Hospital, Fairfield, Australia
A series of organometallic and inorganic drugs has been synthesized at the CSIRO Division of Chemicals and Polymers. The drugs, which are all polyanions of various size, shape and charge are being tested for their activity for the HIV virus in a continuous human T-lymphocyte line (MT21 and in peripheral blood lymphocytes (PBLs). Determinations of drug activity have been carried out at the Fairfield Hospital’s Virology Department. It is important for the drug synthesis programme to develop an understanding of the relationship between polyanion properties and antiviral activity. For this it is essential to establish: (a) whether polyanions enter HIV infected cells, (b) their distribution within these cells, (c) whether this distribution is the same for all polyanions, (d) whether the drugs remain intact (do not dissociate) on entering the cell, (e) the differences between active and inactive drugs of similar structure. Answers to these questions and to others will facilitate the synthesis programme.
1. Introduction
This paper describes work in which the distinctive features of an analytical technique proton induced X-ray emission (PIXE) [l], the versatility of a microscopical instrument (the ion microprobe) [2] and the power of a data handling method (TQSA) [31 combine to handle an otherwise intractable problem in medicine and pharmacology. There is an urgent need to develop satisfactory inhibitors of the human immunodeficiency virus (HIV), causative agent of the acquired immunodeficiency syndrome (AIDS). All currently used drugs have serious side effects. Any antiviral drug is likely to be toxic also to normal cells. The aim in any drug development programme that is based on attacking the virus directly must be to maximise the ratio of toxicity to virus versus toxicity to normal cell and to optimise the delivery of the drug to the site of the virus. A team of chemists at the CSIRO Division of Chemicals and Polymers in Melbourne has developed a line of drugs which are toxic to HIV [4]. These drugs are tested by scientists at the Fairfield Hospital Virology Department for their toxicity to the virus and to normal blood cells. This work alone is very extensive and gives no 1 Permanent
address: Institute of Nuclear Physics, Cracow,
Poland. 0168-583X/93/$06.00
indication as to why one drug may be more effective than another or more toxic to normal cells, while another drug which is closely related chemically may show little effect. What is lacking is any evidence on the relative penetration of the drug into the cells (i.e. are the differences in activity between similar drugs due in part to the variations in their transport efficiency through the cell membranes?) and on their composition within the cell (i.e. has the structure of the applied drug been retained within the cell?). This information would be most valuable for the design of drugs of optimal activity. The drugs are designed to inhibit the replication of HIV in T-lymphocytes. They are tested for this activity and also for their toxicity to healthy cells in a continuous human T-lymhocyte line (MT2 cells) and in peripheral blood lymphocytes (PBLs) from healthy donors. A characteristic of these drugs essential to this investigation is that, apart from the usual light elements found in most drugs, they contain also heavy metal atoms which are not normally seen in cells. These can act as tags to identify the drugs. Unfortunately the drugs are toxic to normal cells and concentrations must therefore be kept below 5 to 200 yg per ml of culture medium, the exact level depending on the drug. The heavy metal concentration within the cell is then well below that measurable with an electron microprobe, but is ideally
0 1993 - Elsevier Science Publishers B.V. All rights reserved
283
M. Cholewa et al. / The use of a scanning proton microprobe in AIDS research
suited to a proton microprobe, because of the extremely low bremsstrahlung level in a PIXE spectrum at the high energy end - that of the so-called heavy metals. What is more, where there are two identifiable tags in the spectrum (from two heavy metals in the drug), a comparison of their ratio within the cell and in the pure drug will indicate whether the drug remains intact within the cell. Finally it is possible to map the distribution of the drug within the cell and its nucleus. This requires a spatial resolution of about 1 micron. The emphasis in this paper is on the techniques and practical details of the microprobe operations involved.
600 E 500 g 400 300
0 2.0
2. Experiment Prior to measurements with the blood cells, PIXE spectra were measured for the pure drugs to be tested. This was partly in order to compare the measured elemental contents with those expected from the design formulae of the synhesised drugs. But the major reason was to establish a quantitative fingerprint for the drug that could be compared with the spectra taken from the cells. In order to avoid unknown energy loss and X-ray absorption in an unknown sample thickness, it was necessary to use either thick or very thin samples of the drugs. Whilst the thick sample effects were in theory calculable, it was decided that quantitative accuracy would be more readily achieved with very thin samples, for which no correction to the spectrum was required. These measurements were more difficult than anticipated, because the drugs were to hard and brittle to section with a microtome and, when solutions of the drugs were allowed to evaporate on nylon foils, the microscopic crystals that formed were not thin enough to totally avoid absorption effects, as evidenced by the nonreproducible spectra obtained. Success was finally achieved by evaporating very weak solutions of
4.0
10.0
6.0 ENERGY (k::)
12.0
Fig. 2. A typical elemental spectrum from a single PBL exposed to 50 yg/ml of GH1532 (KdCo,(H,O>,(PW,O,),] .H,O) drug. The detected concentration of tungsten ( - 10 ppm) in the cell is much above the detection limit of the SPMP, but the level of the drug in the culture medium was a factor of 4 lower than the toxic level.
the drugs in alcohol. Fig. 1 shows a spectrum from one of the drugs, GH1532, whose formula is K,,[Co, (H,O),O'W,O,,~,l . H,O. The lymphocytes were incubated with growth medium containing the drug at various subtoxic levels and this was then removed by resuspending in a buffer of ammonium acetate. Droplets of the cell suspension were placed on nylon foils (mounted on aluminium frames), immediately snap frozen in isopentane cooled by liquid nitrogen and then transferred at liquid nitrogen temperature to a high vacuum vessel in which they were freeze dried. We had previously shown this buffer to be well suited to such work [5], because the ammonium salt evaporates with no trace during the freeze drying process. However, the lymphocytes could not tolerate long exposure to this buffer; so extracellular traces of the drug were removed by repeated centrifu-
2000
,
,
,
,
,
,
,
,
8
100 E
60 60
0 2.0
4.0
6.0 6.0 ENERGY (keV)
Fig. 1. A typical elemental
spectrum
12.0
10.0
from
a GH1532
(K,D[CO,(H~O),(PW,O,~)~I.H~O) drug.
Fig. 3. A typical energy spectrum fr0rn.a single control PBL cell. VI. BIOMEDICAL
284
M. Cholewa et al. / The use of a
scanningproton microprobe in AIDS research
gation in pure growth medium, with a final resuspension in the ammonium acetate (to remove growth medium) requiring exposure for a period of only about 10 min. For a control sample and for samples exposed to a drug at various subtoxic levels, individual lymphocytes were scanned with the microprobe beam of 3 MeV protons. As the approximately 10 micron diameter cells freeze dry on the 1 micron thick nylon foil to discs of approximately 12 micron diameter and 1 micron thickness, there was no need of thick sample corrections but there was need of long exposure to the beam and
Fig. 4. Distribution
of phosphorus,
cobalt
and tungsten
hence good mechanical stability. This was exacerbated by the unusual beam sensitivity of these cells, which would tolerate only 30 pA of beam. A continuously moving, 1 micron diameter beam spot was scanned over the cell for approximately 2 h and all PIXE data were collected by TQSA. Spectra and all elemental maps could be observed during the data collection, so that any errors in identifying and centering healthy cells or any movements could be detected. Fig. 2 shows the spectrum extracted from a cell exposed to the drug GH1532 at a level of 50 ug/ml. The heavy metal elements, Co and W, seen in fig. 1 are again clearly
inside cells when exposed medium.
to a subtoxic
level of GH1.532 drug in the culture
M. Cholewa et al. / The use of a scanning proton microprobe in AIDS research
visible, though the light elements of the drug are swamped by those of the cell. Fig. 3 shows the clear spectrum at high energies for a control cell. If the spectra from many cells are combined, it can be shown that the drug remains intact within the cells, or that the ratio of Co to W at least does not change. The intracellular distributions of these elements can also be mapped and those of P, Co and W are shown in fig. 4. The P tends to concentrate in the cell nucleus and therefore serves to identify this organelle. Hence it is possible to extract spectra for the nucleus and the cytoplasm independently, to measure the intranuclear component of the drug. This requires the accumulation of statistics from a large nuinber of cells.
285
Knowing that these cells were extremely sensitive to irradiation, we examined them with scanning transmission ion microscopy @TIM) [6]. A 2 MeV beam of alpha particles was used in order to maximise the density sensitivity. This beam was focused to less than 200 nm and scanned over a cell. The bright field median image shown in fig. 5a was smoothed with a Gaussian of FWHM 500 nm. In order not to disturb the cell during the PIXE irradiation, the same beam was used, but with 100 pA at 1 micron resolution - a severe exposure which is close to the limit for these cells. Fig. 5b shows a second STIM image of this cell, collected after PIXE irradiation. Quantitative measurements on these two sets of data show that the cell
Fig. 5. STIM maps collected before (A) and after (B) PIXE analysis. Both STIM and PIXE analysis were performed with 2 MeV OL+beams. VI. BIOMEDICAL
286
M. Cholewa et al. / The use of a scanning
shrank in diameter by about 1 micron and in thickness by about 0.2 micron. They also show a sideways movement of about 1 micron. However, the movement and the shrinkages, associated with the loss of light matrix elements [7], would be insufficient to affect the 1 micron resolution of the PIXE data. In order to detect the heavy metal drug elements, it was necessary to use a proton beam and the beam current was also much lower; therefore, for these measurements, the effects would have been much less.
3. Conclusion The measurements reported in this paper are but the first in a series to be carried out in a programme to synthesize drugs with maximum activity against HIV and minimum toxicity to healthy cells. The techniques developed to prepare and handle the blood cells and to make the necessary microprobe measurements have been successful and the heavy metal content of these drugs have proved to be adequate for the identification and quantitative mapping of these drug distributions within their target cells. The measurements are lengthy, in order to collect significant statistics, and the X-ray
proton microprobe in AIDS research collection efficiency is soon to be increased by an order of magnitude with the installation of a new, large area Si(Li) detector.
Acknowledgements We gratefully acknowledge the support of a CSIRO/ University of Melbourne research grant and a Commonwealth AIDS Research Grant.
References [ll T.B. Johansson, R. Akselsson and S.A.E. Johansson, Nucl. Instr. and Meth. 84 (1970) 141. [2] J.A. Cookson, A.T.G. Ferguson and F.D. Pilling, J. Radianal. Chem. 12 (1972) 39. [3] G.J.F. Legge and I. Hammond, J. Microsc. 117 (1979) 201. [4] H. Weigold, G. Holan, S.M. Marcuccio, I.D. Gust and C.J. Birch, Int. Patent Appl., PCT/AU/00280, 28 June 1991. [5] G.L. Allan, J. Camakaris and G.J.F. Legge, Nucl. Instr. and Meth. B54 (1991) 175. [6] J.C. Gverley, R.C. Connolly, G.E. Sieger, J.D. MacDonald and H.W. Lefevre, Nucl. Instr. and Meth. 218 (1983) 43. [7] M. Cholewa, G. Bench, B.J. Kirby and G.J.F. Legge, Nucl. Instr. and Meth. B54 (1991) 101.