- Email: [email protected]
REPLY to Nanomedicine: NMB, 2015; 11:1035
Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1036 – 1037
Reply to Letter to Editor REPLY to Nanomedicine: NMB, 2015; 11:1035 Dear Editor, The observations raised by Somsri Wiwanitkit and Viroj Wiwanitkit concerning our recent report about the encapsulation of metalloporphyrins and their effect on human malaria parasite Plasmodium falciparum 1 highlighted the increasing concern in finding new and safe antimalarial therapies and allow an opportunity to better clarify some aspects of our work. It is well known that one of the effects of chloroquine treatment is to impair the hemozoin formation. In our experiments comparing the hemozoin area in living red blood cells (RBC) infected with P. falciparum, we have confirmed that 20 μM of Zn-PPIX had a similar effect as 1 μM chloroquine on hemozoin growth inhibition after 2 hours incubation. Under these conditions, no relevant differences were observed between Zn-PPIX and chloroquine, however both treatments induced a statistically significant (P b 0.05) decrease on hemozoin area when those groups are compared with a control group treated with DMSO only, as shown in figure and methods section (a full comparison between all groups for this experiment is shown on Table 1). In fact, our work confirmed the results previously obtained by Martiney et al 2 listed as reference 8 of the article. The decrease of hemozoin activity by Zn-PPIX reported by
Table 1 Zn-PPIX vs chloroquine effect on hemozoin inhibition crystal growth in P. falciparum infected RBC. Tukey's multiple comparison test
Mean q difference
Significant: is 95% CI P b 0.05? of diff.
T0 vs DMSO T0 vs chloroquine T0 vs Zn-PPIX DMSO vs chloroquine DMSO vs Zn-PPIX Chloroquine vs Zn-PPIX
− 5.999 21.35 28.05 27.35 34.05 6.699
No No Yes Yes Yes No
1.021 3.635 4.775 4.656 5.796 1.140
− 32.60 to 20.60 − 5.253 to 47.95 1.445 to 54.65 0.7457 to 53.95 7.445 to 60.65 − 19.90 to 33.30
Legend: One-way ANOVA analyses on experiment data of hemozoin inhibition crystal growth in infected red blood cell with Plasmodium falciparum during 2 5hours incubation. Tukey’s multiple comparison test was applied to compare all groups between each other. Groups: T0 = time zero (culture before incubation); DMSO = dimethyl sulfoxide (control incubate with DMSO solvent); Chloroquine = treatment with 1 μM of chloroquine; Zn-PPIX = treatment with 20 μM zinc protoporphyrin. q = q statistic (studentized range statistic). 95% CI of diff. = Confidence interval on the difference between mean on 95% confidence level. Data obtained from 3 different experiments.
nanomedjournal.com
Martiney et al was achieved by using an in vitro assay measured outside living cell. Alves et al 1 reached the same conclusion, however by analyzing an intact infected RBC. Nevertheless, nanomolar concentrations of metalloprotoporphyrins (M-PPIX) in solution were not able to inhibit parasite growth since they failed to access the polymerization site, the food vacuole. Accordingly, encapsulation of M-PPIX in biocompatible polymeric micro and nanocapsules were successfully proposed as an alternative to improve the delivery of those molecules to the parasite's food vacuole, thus enhancing antimalarial activity. The encapsulation of Zn-PPIX in polymeric micro/nanocapsules reduced 80 times the IC50 value as compared with non-encapsulated Zn-PPIX in solution. This reduction of IC50 from micromolar to nanomolar concentration range should diminish the possibility of toxic effects as a consequence of the lower doses. In addition, the polymeric micro/nanocapsules also lowered the toxicity of Zn-PPIX as demonstrated by our experiments using HEK cells. Ju et al 3 reported a decrease in the toxicity when CdSe/ZnS quantum dots (Qdots) were coated with polyethylene glycol (PEG), as confirmed by diminished DNA damage and ROS generation as compared to the original Qdots. In fact, several researchers have reported lower cytotoxicity for nanomaterials and several substances with pharmacological activity after coating with a polymeric layer. We share the same concerns relative to safety and would like to thank for the suggestion to investigate further the toxicity of encapsulated Zn-PPIX. The results of a detailed study will be published as soon as possible.
Eduardo Alves, PhD Bernardo Iglesias, PhD Daiana Deda, PhD Alexandre Budu, PhD Tiago Matias, MS Vania Bueno, PhD Fernando Maluf, BS Rafael Guido, PhD Glaucius Oliva, PhD Luiz Catalani, PhD Koiti Araki, PhD Celia Regina da Silva Garcia, PhD ⁎ Instituto de Biociencias, Universidade de Sào Paulo Sào Paulo, Brazil ⁎Corresponding author E-mail address: [email protected]
http://dx.doi.org/10.1016/j.nano.2015.01.015
1549-9634/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Alves E, et al, REPLY to MS#JN2014639. Nanomedicine: NBM 2015;11:1036-1037, http://dx.doi.org/10.1016/ j.nano.2015.01.015
Reply to Letter to Editor / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1036–1037
References 1. Alves E, Iglesias BA, Deda DK, Budu A, Matias TA, Bueno VB, et al. Encapsulation of metalloporphyrins improves their capacity to block the viability of the human malaria parasite Plasmodium falciparum. Nanomedicine 2014 http://dx.doi.org/10.1016/j.nano.2014.09.018 [pii: S15499634(14)00550-4, [Epub ahead of print].
1037
2. Martiney JA, Cerami A, Slater AF. Inhibition of hemozoin formation in Plasmodium falciparum trophozoite extracts by heme analogs: possible implication in the resistance to malaria conferred by the beta-thalassemia trait. Mol Med 1996;2(2):236-46. 3. Ju L, Zhang G, Zhang C, Sun L, Jiang Y, Yan C, et al. Quantum dotrelated genotoxicity perturbation can be attenuated by PEG encapsulation. Mutat Res 2013;753(1):54-64.
Reply to Letter to Editor REPLY to Nanomedicine: NMB, 2015; 11:1035 Dear Editor, The observations raised by Somsri Wiwanitkit and Viroj Wiwanitkit concerning our recent report about the encapsulation of metalloporphyrins and their effect on human malaria parasite Plasmodium falciparum 1 highlighted the increasing concern in finding new and safe antimalarial therapies and allow an opportunity to better clarify some aspects of our work. It is well known that one of the effects of chloroquine treatment is to impair the hemozoin formation. In our experiments comparing the hemozoin area in living red blood cells (RBC) infected with P. falciparum, we have confirmed that 20 μM of Zn-PPIX had a similar effect as 1 μM chloroquine on hemozoin growth inhibition after 2 hours incubation. Under these conditions, no relevant differences were observed between Zn-PPIX and chloroquine, however both treatments induced a statistically significant (P b 0.05) decrease on hemozoin area when those groups are compared with a control group treated with DMSO only, as shown in figure and methods section (a full comparison between all groups for this experiment is shown on Table 1). In fact, our work confirmed the results previously obtained by Martiney et al 2 listed as reference 8 of the article. The decrease of hemozoin activity by Zn-PPIX reported by
Table 1 Zn-PPIX vs chloroquine effect on hemozoin inhibition crystal growth in P. falciparum infected RBC. Tukey's multiple comparison test
Mean q difference
Significant: is 95% CI P b 0.05? of diff.
T0 vs DMSO T0 vs chloroquine T0 vs Zn-PPIX DMSO vs chloroquine DMSO vs Zn-PPIX Chloroquine vs Zn-PPIX
− 5.999 21.35 28.05 27.35 34.05 6.699
No No Yes Yes Yes No
1.021 3.635 4.775 4.656 5.796 1.140
− 32.60 to 20.60 − 5.253 to 47.95 1.445 to 54.65 0.7457 to 53.95 7.445 to 60.65 − 19.90 to 33.30
Legend: One-way ANOVA analyses on experiment data of hemozoin inhibition crystal growth in infected red blood cell with Plasmodium falciparum during 2 5hours incubation. Tukey’s multiple comparison test was applied to compare all groups between each other. Groups: T0 = time zero (culture before incubation); DMSO = dimethyl sulfoxide (control incubate with DMSO solvent); Chloroquine = treatment with 1 μM of chloroquine; Zn-PPIX = treatment with 20 μM zinc protoporphyrin. q = q statistic (studentized range statistic). 95% CI of diff. = Confidence interval on the difference between mean on 95% confidence level. Data obtained from 3 different experiments.
nanomedjournal.com
Martiney et al was achieved by using an in vitro assay measured outside living cell. Alves et al 1 reached the same conclusion, however by analyzing an intact infected RBC. Nevertheless, nanomolar concentrations of metalloprotoporphyrins (M-PPIX) in solution were not able to inhibit parasite growth since they failed to access the polymerization site, the food vacuole. Accordingly, encapsulation of M-PPIX in biocompatible polymeric micro and nanocapsules were successfully proposed as an alternative to improve the delivery of those molecules to the parasite's food vacuole, thus enhancing antimalarial activity. The encapsulation of Zn-PPIX in polymeric micro/nanocapsules reduced 80 times the IC50 value as compared with non-encapsulated Zn-PPIX in solution. This reduction of IC50 from micromolar to nanomolar concentration range should diminish the possibility of toxic effects as a consequence of the lower doses. In addition, the polymeric micro/nanocapsules also lowered the toxicity of Zn-PPIX as demonstrated by our experiments using HEK cells. Ju et al 3 reported a decrease in the toxicity when CdSe/ZnS quantum dots (Qdots) were coated with polyethylene glycol (PEG), as confirmed by diminished DNA damage and ROS generation as compared to the original Qdots. In fact, several researchers have reported lower cytotoxicity for nanomaterials and several substances with pharmacological activity after coating with a polymeric layer. We share the same concerns relative to safety and would like to thank for the suggestion to investigate further the toxicity of encapsulated Zn-PPIX. The results of a detailed study will be published as soon as possible.
Eduardo Alves, PhD Bernardo Iglesias, PhD Daiana Deda, PhD Alexandre Budu, PhD Tiago Matias, MS Vania Bueno, PhD Fernando Maluf, BS Rafael Guido, PhD Glaucius Oliva, PhD Luiz Catalani, PhD Koiti Araki, PhD Celia Regina da Silva Garcia, PhD ⁎ Instituto de Biociencias, Universidade de Sào Paulo Sào Paulo, Brazil ⁎Corresponding author E-mail address: [email protected]
http://dx.doi.org/10.1016/j.nano.2015.01.015
1549-9634/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Alves E, et al, REPLY to MS#JN2014639. Nanomedicine: NBM 2015;11:1036-1037, http://dx.doi.org/10.1016/ j.nano.2015.01.015
Reply to Letter to Editor / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1036–1037
References 1. Alves E, Iglesias BA, Deda DK, Budu A, Matias TA, Bueno VB, et al. Encapsulation of metalloporphyrins improves their capacity to block the viability of the human malaria parasite Plasmodium falciparum. Nanomedicine 2014 http://dx.doi.org/10.1016/j.nano.2014.09.018 [pii: S15499634(14)00550-4, [Epub ahead of print].
1037
2. Martiney JA, Cerami A, Slater AF. Inhibition of hemozoin formation in Plasmodium falciparum trophozoite extracts by heme analogs: possible implication in the resistance to malaria conferred by the beta-thalassemia trait. Mol Med 1996;2(2):236-46. 3. Ju L, Zhang G, Zhang C, Sun L, Jiang Y, Yan C, et al. Quantum dotrelated genotoxicity perturbation can be attenuated by PEG encapsulation. Mutat Res 2013;753(1):54-64.