**5. New treatment proposals**

Due to intense collateral effects, lots of people who are infected with leishmaniases refuse treatment, and there is the need to new alternative treatments. One of the new possibilities in study nowadays uses natural rubber (NR) membranes with metallic nanoparticles (MNPs) such as silver and gold (SNPs and GNPs, respectively). NR membranes are used as an active support to the MNPs reduction process using precursor salts to obtain metallic particles, and that means that the NR membrane works as a reducing agent as well as a stabilizer for MNPs.

When a promastigote population of *Leishmania braziliensis* is in the presence of pure natural rubber membranes, there is a significant increase in the log phase of the cells, constituted by a large number of cells at the division process. In the 27th hour after inoculation, the parasite colony remains stable, without large variations in the population cells. In this phase, called stationary phase, the promastigote cells start morphophysiological transformations known as metacyclogenesis. With the end of the stationary phase, an environmental saturation happens with cell waste and lack of nutrients, resulting in the death of cells, phase known as fall.

This decrease could be explained by the natural dynamics of promastigotes in the culture medium. It is expected that after 150 hours, this decrease in the population number occurs because of nutritional deficit and saturation of the cellular environment with cellular excreta generated during the period of growth log and stationary phase, but the presence of nano‐ particles anticipates the drop in the number of the parasites, probably because these metal components stop the absorption of nutrients of the environment through permeases and transplasmalema transport channels.

However, when promastigotes interact with natural rubber membranes impregnated with silver nanoparticles, a stretch of the first metabolic phase is seen, only with a non-significant growth. In figure 5 is possible to see that the decrease in *L. braziliensis* is proportional to the increase of silver concentrations in the NR membrane.

Silver nanoparticles toxicity is well known, and although death of the promastigotes may occur, it is believed that changes in the physiological maturation process, such as drastic changes in the surface glycoproteins, happen to the microorganism cell. In that way, the NR membranes with silver nanoparticles when put in the protozoa medium is capable of sepa‐ rating the cells without killing the whole population, eliminating only the ones which did not finalized their metacyclogenesis and are highly infective to the hosts (Figure 6). Immature cells that are not able to generate a disease are kept intact, and this process is what generates an important biomass to a future study focus a vaccine against this disease.

**Figure 5.** Populational growth of *Leishmania braziliensis* promastigotes in contact with natural rubber membranes and natural rubber membranes impregnated with silver nanoparticles using *in situ* reduction.

Natural rubber membranes containing GNPs (NR/Au) also decrease the population growth rate, showing a lower index of living promastigotes (attached to the membrane surface) depending on the amount of nanoparticles deposited in the membrane surface. Formation and growth of the GNPs within the NR membranes, by reduction of Au3+, was monitored following the Plasmon absorption band. Reducing agents for GNPs formation are likely to be the carboxylic functional groups present in the NR. Nanoparticle, which produces an increase of the intensity of the distinctive plasmon absorption band of GNPs centered at approximately 560 nm, as shown in Figure 7 [33].

Nanoparticle Technology: An alternative approach for Leishmaniasis Treatment http://dx.doi.org/10.5772/57283 385

generated during the period of growth log and stationary phase, but the presence of nano‐ particles anticipates the drop in the number of the parasites, probably because these metal components stop the absorption of nutrients of the environment through permeases and

However, when promastigotes interact with natural rubber membranes impregnated with silver nanoparticles, a stretch of the first metabolic phase is seen, only with a non-significant growth. In figure 5 is possible to see that the decrease in *L. braziliensis* is proportional to the

Silver nanoparticles toxicity is well known, and although death of the promastigotes may occur, it is believed that changes in the physiological maturation process, such as drastic changes in the surface glycoproteins, happen to the microorganism cell. In that way, the NR membranes with silver nanoparticles when put in the protozoa medium is capable of sepa‐ rating the cells without killing the whole population, eliminating only the ones which did not finalized their metacyclogenesis and are highly infective to the hosts (Figure 6). Immature cells that are not able to generate a disease are kept intact, and this process is what generates an

**Figure 5.** Populational growth of *Leishmania braziliensis* promastigotes in contact with natural rubber membranes

Natural rubber membranes containing GNPs (NR/Au) also decrease the population growth rate, showing a lower index of living promastigotes (attached to the membrane surface) depending on the amount of nanoparticles deposited in the membrane surface. Formation and growth of the GNPs within the NR membranes, by reduction of Au3+, was monitored following the Plasmon absorption band. Reducing agents for GNPs formation are likely to be the carboxylic functional groups present in the NR. Nanoparticle, which produces an increase of the intensity of the distinctive plasmon absorption band of GNPs centered at approximately

and natural rubber membranes impregnated with silver nanoparticles using *in situ* reduction.

560 nm, as shown in Figure 7 [33].

transplasmalema transport channels.

384 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

increase of silver concentrations in the NR membrane.

important biomass to a future study focus a vaccine against this disease.

**Figure 6.** Separation Process of *Leishmania* in contact with NR membranes with silver (Ag) nanoparticles; (a) and (b) NR/Ag membranas in culture medium; (c) *Leishmania braziliensis* in contact to NR/Ag membranes; (d) Separated pro‐ tozoa attached to NR/Ag membranes.

**Figure 7.** UV-Vis spectroscopy of natural rubber/gold nanoparticles prepared at different gold reduction times, com‐ pared to raw natural rubber membrane (preparation of the membrane annealed at 65 °C).

Analyses by scanning electron microscopy (ESEM) were performed for natural rubber membranes with gold nanoparticles, obtained at a reduction time of 30 min (Figure 8). Apparently, there is a homogenous distribution of nanoparticle occupying the entire surface of the membrane, but by amplifying the images, one can observe the formation of many nanoparticles in sites, and among them, nanoparticles dispersed on the surface of the poly‐ meric matrix. In Figure 8 (d) the small spherical nanoparticles can be seen agglomerated over reduction sites, forming aggregates at an exorbitant amount. Nanoparticles sizes were measured and are very similar, with an average of approximately 48 nm. These results are in agreement with the analysis by UV-Vis spectroscopy.

**Figure 8.** SEM analysis of NR/Au membrane obtained at 30 minutes of reduction. Magnification of (a) 200 times; (b) 1,000 times; (c) 20,000 times; (d) 100,000 times.

Natural rubber membranes, with colloidal incorporation of gold nanoparticles, were synthe‐ sized by *in situ* method (NR/Au) at different reduction times, in order to evaluate the physio‐ logical behavior of *L. braziliensis* promastigotes cultured in axenic medium, in a colony of seven weeks old, evaluating the increase kinetics in relation to the permanence time of the mem‐ branes in the culture medium as a function of reduction time and temperature of preparation of natural rubber membranes.

It was observed that the inclusion of NR/Au membrane in culture generates a delayed onset of growth in culture until about 24 hours, with a reduction in the development population proportional to the time reduction of the gold nanoparticles. It is also noted that the NR/Au 120 min. membrane presents the best results with regard to growth inhibition of promastigote population in culture medium. NR/Au 30 min membrane has a sharper decline in curve kinetics attributable to death of promastigotes from 120 hours. Upon analysis completion of growth medium, the membranes prepared for times of 30, 60 and 120 minutes showed similar results, with the number of promastigotes in solution smaller than inserted into the initial population.

Analyses by scanning electron microscopy (ESEM) were performed for natural rubber membranes with gold nanoparticles, obtained at a reduction time of 30 min (Figure 8). Apparently, there is a homogenous distribution of nanoparticle occupying the entire surface of the membrane, but by amplifying the images, one can observe the formation of many nanoparticles in sites, and among them, nanoparticles dispersed on the surface of the poly‐ meric matrix. In Figure 8 (d) the small spherical nanoparticles can be seen agglomerated over reduction sites, forming aggregates at an exorbitant amount. Nanoparticles sizes were measured and are very similar, with an average of approximately 48 nm. These results are in

**Figure 8.** SEM analysis of NR/Au membrane obtained at 30 minutes of reduction. Magnification of (a) 200 times; (b)

Natural rubber membranes, with colloidal incorporation of gold nanoparticles, were synthe‐ sized by *in situ* method (NR/Au) at different reduction times, in order to evaluate the physio‐ logical behavior of *L. braziliensis* promastigotes cultured in axenic medium, in a colony of seven weeks old, evaluating the increase kinetics in relation to the permanence time of the mem‐ branes in the culture medium as a function of reduction time and temperature of preparation

It was observed that the inclusion of NR/Au membrane in culture generates a delayed onset of growth in culture until about 24 hours, with a reduction in the development population proportional to the time reduction of the gold nanoparticles. It is also noted that the NR/Au

agreement with the analysis by UV-Vis spectroscopy.

386 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

1,000 times; (c) 20,000 times; (d) 100,000 times.

of natural rubber membranes.

According to the results the best allowed reduction time is 120 minutes, due to its greater population growth inhibition, and was then assessed the influence of the thermal treatment by the casting preparation method of membrane natural rubber annealed at 60, 80 and 120 °C, and then used as substrates for the reduction of nanoparticles. Results obtained for the kinetics of increase in solution are shown in Figure 9.

**Figure 9.** Analysis of the kinetics of increase of promastigotes in solution in the presence of natural rubber mem‐ branes prepared by the casting method, at temperatures of 65, 80 and 120 °C, and then incorporated gold nanoparti‐ cles to the time reduction of 120 minutes.

It is observed that with increasing of thermal treatment temperature in the preparation of NR membranes the population growth factor in culture decreases, reaching scores lower than 2x105 promastigotes (less than half of the inoculum) to membranes prepared at 120 °C at a reduced nanoparticles to 120 minutes. It is known from promastigotes reactivity with carbox‐ ylic and nitrogenated groups, the same reactive groups and assigned to the reduction of nanoparticles. Higher the temperature of thermal treatment used in the preparation of NR membranes, smaller the amount of nanoparticles embedded in the membrane surface, attributed to the fact that there is further degradation of the active components of the mem‐ brane with increasing temperature. Thus, it is attributed the decrease in population growth in solution to generate more reactive nanoparticles or selective proteins contained in the culture medium, inhibiting the development of promastigotes, and may also be linked to decreasing of active cores on the membrane surface, or even liberation of radicals in the culture medium attributed to membrane preparation temperature, but the level of promastigotes in culture medium still remains considerable, so that in an attempt to develop a smart skin bandage incorporated into methylene blue molecules on Au particles to photodynamic treatment, due to the drug photosensitivity.

Membranes of pure natural rubber and with incorporation of gold nanoparticles and methyl‐ ene blue were inserted in the culture medium and the behavior of *L. braziliensis* promastigotes were evaluated through the population growth kinetics and morphophysiological analysis. Results regarding to population growth obtained for protozoan eluted to membranes prepared at 120 °C of thermal treatment are presented in Figure 10.

**Figure 10.** Molecular spectroscopy in UV-Vis region of natural rubber membranes with gold nanoparticles for reduc‐ tion time of 120 min and functionalization with methylene blue molecules, compared to pure natural rubber mem‐ branes.

To improve the studies, blue methylene was incorporated to the NR membrane. This incor‐ poration does not show toxic interaction with promastigotes, since the population growth usually develops when inserted NR/MB membranes. Moreover, when irradiated culture media containing BM molecules, a deficit in the population growth is observed. For NR/MB membranes irradiated with blue light total elimination of promastigotes is observed in about 120 hours, whereas when MB molecules deposited on gold nanoparticles this time decreases to about 60 hours. This fact is attributed to the amplification effect of surface commonly evaluated by micro-Raman spectroscopy, so that the metallic particles act as sites for amplifi‐ cation of energy absorbed by the anchored molecules.

The electrons assume an excited energy state, and during the release of the absorbed energy active radicals are generated, with interaction with the environment where are MB molecules. In the first instance, electrons from MB molecules, when returning to the ground state emit enough energy to excite the oxygen present in the medium converting to superoxides or peroxides radials. This transfer may occur also by interaction with hydrogen atoms present in other molecules to form radicals, e.g. hydroxyl, also with potential toxic to cells, or as in this case, the promastigotes. If the emission energy is derived from a transition to a triplet state of the photosensitizer, one can obtain the formation of a singlet oxygen molecule and an oxidizing agent more toxic to cells [34-38].
