Photodynamic decontamination of blood for transfusion

E.Ben-Hur, H.Margolis-Nunno, P.Gottlieb, S.Lustigman and B.Horowitz

New York Blood Center, 310 E.67th Street, New York, NY 10021

ABSTRACT

Currently transfused cellular components of blood are not available in a sterile form and carry a small risk of 
transmitting viral and parasite diseases. Using phthalocyanines and red light, lipid enveloped viruses, e.g. HIV-1, 
can be inactivated in red blood cell concentrates (RBCC). Under conditions leading to virus sterilization the 
blood borne parasites Trypanosoma cruzi (Chagas disease) and Plasmodium falciparum (malaria) could be 
eliminated to undetectable levels ( > 4 log10 kill). RBC damage during treatment could be avoided by increasing 
the light fluence rate to 80 mW/cm2, and by including the free radical scavenger glutathione and the vitamin E 
derivative Trolox during light exposure. Similar sterilization of platelet concentrates was achieved with the 
psoralen derivative AMT and UVA light. Platelet damage due to PUVA treatment was avoided by including the 
plant flavonoid rutin during irradiation. It is concluded that elimination of the risk of'transmitting pathogens 
during blood transfusion is feasible with photochemical treatments.


2. Introduction

Transmission of pathogenic viruses by blood transfusion has been reduced in recent years by serological 
screening for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). 
However, absolute safety has not been achieved and the risk of HBV, HCV AND HIV-1 transmission in the USA 
with a single blood unit has been estimated at 0.0005%, 0.03% and 0.0005%, respectively.' Patients who receive 
a large number of red blood cell concentrates (RBCC) are at a much higher risk of virus transmission. Other 
viruses of concern in patients with compromised immune systems are cytomegalovirus (CMV) and parvovirus. 
In addition, transmission of parasitic infections are of much concern in developing countries. These inciude 
Chagas disease, caused by Trypanosoma cruzi and malaria, caused by Plasmodium sp. (T.cruzi is endemic in 
Latin America and malaria is endemic in Africa and some parts of Asia). In the USA, T.cruzi infection among 
immigrants from Latin America is about 5%2 and cases of transfusion-transmitted Chagas disease have been 
reported.3

Sterilization appears to be the best way to ensure a very high level of safety in transfusion o f blood and its 
components. Currently, all blood products are available in sterilized forms with the exception of red cell and 
platelet concentrates. Sterilization of cellular blood components presents a unique challenge because cell 
structure and function are disrupted more easily than those of individual proteins. Various approaches have been 
taken for virus sterilization of RBC and platelets4, however, favorable results were obtained only with 
photodynamic treatment (PDT)5. As a result, almost all the efforts are now focused on this approach.

In our work, for the treatment of platelets we chose psoralens which target nucleic acids and are activated by 
UVA light. This approach has been adopted also by other workers and will be discussed only briefly. For 
sterilizing RBC the use of UVA is not effective because of the strong absorption by hemoglobin. We therefore 
selected for this purpose the phthalocyanines (Pc) which are activated by light in the far red (650-700 nm).

3. STERILIZATION OF PLATELET CONCENTRATES

Psoralen and its derivatives (Fig.1) are heterocyclic, planar compounds that are found in many plants. Psoralens 
intercalate between the bases of nucleic acids and upon exposure to UVA light can form monoadducts and 
crosslinks with pyrimidines.6 The ability of psoralen to target nucleic acids is an obvious advantage for 
decontamination of platelets, which lack a nucleus. However, because psoralens can produce singlet oxygen, 
light exposure is usually done in the absence of oxygen to avoid damage to platelets.7

Our approach for sterilization of platelet concentrates has been to use a derivative of 4,5',8-trimethylpsoralen 
(TMP) containing an amine hydrochloride group (AMT). Due to its cationic nature AMT has a very high affinity 
to nucleic acids and is much more effective in killing single-stranded RNA viruses than either TMP or 8-MOP. 
To avoid photodynamic reactions, rather than removing oxygen we included scavengers of reactive oxygen 
species such as mannitol and plant fiavonoids.89 Under our current treatment conditions (50 pg/ml AMT, 0.35 
mM rutin and 38 J/cm2 UVA) complete inactivation ( > 6 logo) of cell-free and cell-associated vesicular 
stomatitis virus (VSV) is obtained without compromising platelet structure and function. This treatment resulted 
also in > 5 log10 kill of T.cruzi.

4. STERILIZATION OF RED CELL CONCENTRATES

4.1 Virucidal action of phthalocyanines

Phthalocyanines are porphyrin-like synthetic pigments with a macrocycle made up of four isonindole units 
linked by nitrogen atoms (Fig.2).

Phthalocyanines have an intense absorption in the far red which is ideal for PDT of cancer and sterilization of 
red cells, as there is little absorption by hemoglobin at these wavelengths. When substituted with diamagnetic 
metals phthalocyanines have long lived excited triplet state and can generate singlet oxygen at a high quantum 
yield.'  However, the relative contributions of type i and type il photodynarnic reactions in biological systems is 
not known and appears to vary from system to system. For a recent review of the photobiology of 
phthalocyanines see ref. 1 1 .

Lipid enveloped viruses can be inactivated by phthalocyanines. The efficacy of virus kill depends on the 
substituents on the Pc macrocycle.'2~'5 However, a clear structure-activity relationship has not emerged so far.'5 
Concomitantly with virus inactivation, Pc-PDT can cause RBC damage. Again, the dependence of RBC damage 
on a particular structural feature of Pc has not been established, although metallo PcS4 appear to have the least 
photohemolytic activity.'5 Activity is usually correlated with binding of the dye to RBC.'5 16

Among all the phthalocyanines tested so far, the most potent with regard to virus inactivation are the cationic 
silicon phthalocyanine, Pc5, and its neutral analog, Pc4. The latter is also highly effective in PDT of cancer.'7 
Because Pc4 caused less RBC damage than Pc5 it is currently our leading candidate for sterilization of RBCC. 
As will be shown later, Pc4 is also more effective than Pc5 in killing blood-borne parasites.

4.2 Type I quenchers for enhanced specificity

A certain amount of selectivity of virus inactivation without compromising RBC integrity can be obtained by 
seiecting the appropriate phthalocyanine. This selectivity, however, is not absolute. One way to enhance 
specificity is by adding scavengers of free radicals produced by type I photodynamic reactions. Such quenchers, 
e.g. mannitol and glutathione (GSH), do not

affect virus inactivation by phthalocyanines and red light since virus kill appears to proceed via type 11 
reaction.'8 However, RBC damage is mediated by both type I and type 11 mechanisms.'8'9 As a result, including 
type I quenchers during virus inactivation in RBC enhances the specificity of the reaction. Thus, the use of 
mannitol and GSH preserves in vivo circulatory survival of rabbit RBC treated with AlPcS4 under virucidal 
conditions.'8 Inclusion of GSH has the added advantage of eliminating IgG binding to treated RBC. This is 
important for blood banking purposes since IgG binding prevents blood typing. Inhibition of such binding 
induced by PDT can be prevented only by SH containing compounds.

Another type I quencher found to be useful in enhancing the specificity of virus inactivation is vitamin E. Thus, 
=-tocopherol succinate inhibits AlPcS4-induced photohemolysis20 and Trolox, a water-soluble vitamin E 
derivative was found in addition to prevent PDT-induced reduction of RBC negative surface charge and K+ 
leakage without affecting the rate of VSV kill.2'.

4.3 Enhanced specificity at higher fluence rates

Cells are able to deal with reactive oxygen species by various mechanisms such as quenchers and antioxidant 
enzymes.22 Therefore, it is expected that when photodynamic treatment is delivered at a reduced fluence rate 
the cells will be able to tolerate it better. This indeed has been shown for mammalian cells photosensitized with 
AlPcSn23 and HPD.24 Viruses are devoid of protective mechanisms and their inactivation rate is therefore not 
expected to be affected by changes in light fluence rate. The latter assumption was indeed found tolbe true. 
However, in contrast to expectations, the extent of RBC damage decreased with increasing fluence rate.25 The 
most trivial explanation for this is that oxygen becomes rate limiting for the photodynamic reactions at high 
fluence rates. This possibility has been ruled ouFt because the effect was not reduced when exposure was carried 
out in oxygen-saturated rather than airsaturated solutions.25

Other possible explanations for the fluence rate effect can be invoked and are based on an enhancement of the 
quenching of reactive species at high fluence rates. For example, 102 can form dimoles, (1 2)2s the 
concentration of which should be proportional to the square of the light fluence rate.26 However, when 
irradiation was done in D2O the fluence rate effect remained unchanged, arguing against dimol contribution to 
the basic observations. Another possibility is quenching of 102 by sensitizer molecules in their triplet excited 
states.27 This mechanism should be characterized by a decreasing yield  f 1 2 with increasing fluence rate. The 
observation of such a mechanism should be facilitated at high sensitizer concentration. However, reducing the 
concentration of phthalocyanine by 4-fold did not affect the fluence rate effect.25 This also argues against 
recombination of other reactive oxygen species (e.g. OH radicals) as a possible explanation.

Whatever the mechanism is for the fluence rate effect in RBC, its absence for virus kill has an obvious 
advantage for photochemical sterilization. The use of high fluence rates at 80 mW/cm2 and above would 
minimize RBC damage and allow higher- light fluences to be used in the process.

4.4 Inactivation of parasites in RBC

The only effective chemoprophylaxis against T.cruzi available in endemic areas is gentian violet, which is used 
at a concentration of 0.6 mM in blood to be transfused.23 A good trypanocidal effect by the dye takes 24 h at 4  
C and is effective against all parasite stages. While there are no major deleterious effects on RBC and platelets, 
the blood in recipients turns a purple color which stains the skin and mucosa, and in rodents there is a 
carcinogenic effect. Thousands of recipients have received treated blood without any apparent side effects but 
controlled studies are not available to better understand possible toxic effects of gentian violet.

There are currently no methods for inactivation of P.falciparum in RBCC. Merocyanine 540 has been reported to 
reduce the concentration of parasitized RBC by 3 log10 when exposed to light.29 However, because there is 
significant overlap between the absorption spectrum of merocyanine 540 and that of hemoglobin, this dye is not 
suitable for use in RBCC.

Our efforts to inactivate T.cruzi and P.falciparum in RBCC were quite successful.30 The results are summarized 
in Table 1. Of all the phthalocyanines tested, Pc4 appears to be the most effective, reducing parasites below 
detection level already after 10 min light exposure. For comparison, complete virus kill with Pc4 requires 20-30 
min light exposure under the same conditions.

5. CONCLUSIONS

Virus and parasite sterilization of RBCC and platelet concentrates using Pc-PDT and PUVA, respectively, 
appears to be feasible. Nonetheless, important questions remain. The efficacy with respect to sterilization of 
HCV and HIV (the latter in blood from AIDS patients) need to be demonstrated and the treated cells need to be 
evaluated with respect to potential neoimmunogenicity, circulatory survival and half-life, and ultimately 
clinically. Even though a step to remove the additives after light exposure is being implemented, appropriate 
toxicological studies have to be done to ensure safety to the recipient.

6. ACKNOWLEDGEMENTS

Thanks are due to Prof. M.E.Kenney for supplying Pc4, Pc5 and other phthalocyanines for this study. Work was 
supported in part by award No. R01 -HL41221 from the National Heart, Lung and Blood Institute.

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