Under current development is Nanosense, a medication designed for the nonsurgical treatment of superficial cancer via pulsed laser ablation of nanoparticles (PLAN). Nanosens is a stable dispersion of phthalocyanine zinc nanoparticles in a physiological solution. The technology for obtaining stable dispersions of zinc phthalocyanine ensures a shelf life of over two years. This article presents preliminary data from preclinical studies of the Nanosens formulation. Results were obtained on the effectiveness, pharmacokinetics, and biodistribution of the Nanosens drug in mice, as well as toxicity and allergy studies. When tested at therapeutic doses (1 TD = 7 mg/kg) ranging from 1 to 10 TD for acute toxicity and from 1 to 30 TD for chronic toxicity, no signs of intoxication leading to death were observed in animals. However, at the maximum dose, individual organs and tissues exhibited a blue coloration.

Nanosense, nonsurgical, treatment, PLAN, phthalocyanine, photodynamic, tumor, nanotechnology, toxicity, microexplosive, ablation

Today, some of the concerns in public health revolve around resistance and cancer, leading to the loss of more than 15 million lives annually. This highlights a decline in the effectiveness of antibiotics and chemotherapy treatments. One proposed solution is the application of nanotechnology.

One of the most significant achievements of recent years is the study of methods for obtaining the properties of nanoparticles, quantum dots, and other nano-sized objects. The number of publications on this topic is rapidly increasing, with the first examples of commercial applications of nanomaterials emerging.

Since this field of science began to be thoroughly explored relatively recently, there is still no clear classification of nanoparticles. However, three distinct groups of nanoparticles can be identified: metal nanoparticles, nanoparticles of inorganic compounds, and organic nanoparticles. The first two groups have been studied in sufficient detail, with extensive literature on their synthesis, optical and semiconductor properties, and research methods. In contrast, organic nanoparticles are much less studied, with only a few articles focusing on the synthesis and investigation of nanoparticles of phthalocyanine dyes and some other organic compounds.

Based on literature analysis, the question of the sizes of organic nanoparticles and methods of their measurement, as well as the influence of the size parameter on the manifestation of photo-physical effects, remains insufficiently explored in this area of knowledge. Research methods in electron microscopy and small-angle X-ray scattering are not well suited here: firstly, organic nanoparticles are largely transparent to electrons and X-rays, and secondly, strong impacts, such as electron beam exposure, contribute to their destruction. It becomes evident that nanoparticles are better studied in the matrix in which they were synthesized, and non-destructive methods of control should be used for their investigation (Gulbinas et al. 1994; Wang et al. 1999; Akimov et al. 2003; Denisyuk and Kamanina 2004).

The sizes of crystalline nanoparticles appear to stay constant without any changes over time. For instance, the size of Mg phthalocyanine nanoparticles is 2 nm in the β form. 2.5 nm in the X form, aligning with the sizes of cells within the crystalline lattice of their respective polymorphic forms. It is proposed that each nanocrystal consists of two cells from the crystalline lattice, ensuring its stability. On the other hand, amorphous modification nanoparticles are less stable; their sizes tend to increase from 12 to 22 nm during storage, eventually leading to colloid precipitation (Butyanov et al. 2005).

A Russian scientist (Kogan et al. 2004; Pankratov et al. 2014) has discovered that photoactive aluminum phthalocyanine nanoparticles, zinc phthalocyanine, and metal-free phthalocyanine, under the influence of pulsed laser radiation, can form molecular forms that act as photosensitizers. Phthalocyanines are organic dyes that are inert, non-toxic, and insoluble in organic solvents and water. It has been found that phthalocyanine nanoparticles can be used as effective “pro-photosensitizers” for the treatment of malignant tumors using photodynamic therapy. Activation of a photoactive nanoparticle into a photoactive molecular photosensitizer occurs locally within the tumor nods by irradiating with powerful laser pulses. During this process, the nanoparticle explodes, exhibiting photosensitizer properties. Such “generated” photosensitizer directly within the tumor can be used to conduct conventional photodynamic therapy.

The main drawback of conventional photosensitizers is their cutaneous phototoxicity, manifested by increased photosensitivity of the skin and the need for protection from sunlight. In this case, even with repeated administration of AIPc, ZnPc, and H2Pc nanoparticles, there was no accumulation of their photoactive form in the skin, which could contribute to the development of skin phototoxicity.

As a result of the conducted research, effective protocols for photodynamic therapy using a medical preparation (Nanosense) containing zinc photalocyanine nanoparticles in the form of an aqueous suspension have been identified.

The pharmaceutical preparation Nanosense is a bright blue liquid—a stabilized nanodispersion of zinc phthalocyanine (see Fig. 1) in physiological saline. The content of the active substance in the preparation is 2 mg/mL. The average particle size of zinc phthalocyanine is 150–250 nm. The shelf life of the preparation is at least two years.

Figure 1. 

Zinc Phthalocyanine.

Nanosense is the first pharmaceutical preparation designed for the treatment of skin cancer using the method of pulse laser ablation nanoparticles (PLAN).

The study of the antitumor effectiveness of the Nanosens was carried out using the PLAN method against transplantable tumors of mice of various histogenesis.

Equipment used in the study: as a source of powerful pulsed laser radiation, we used a pulsed ruby laser (λ=694 nm; pulse duration 40 ms), operating in the modulation mode (Pockels cell) of the resonator Q factor (irradiation parameters are presented in the designs of the corresponding studies). Manufacturer: FSUE “SSC “NIOPIK”. The PLAN method was developed jointly by scientists from FSUE “GNC “NIOPIK” FSBI “MNIOI named after P.A. Herzen” (Kogan et al. 2004, 2006, 2008, 2010, 2011a, 2011b; Andreeva et al. 2012; Pankratov et al. 2014). It is protected by three patents (RF Patent 2008; RF Patent 2010; Patent RU 2017). The technology for producing the Nanosense preparation was developed at the FSUE “GNC “NIOPIK”.

The methodology for studying the antitumor effectiveness was carried out depending on doses of the drug, time interval between drug administration and pulsing irradiation, parameters of pulsed laser radiation, energy density per pulse, and number of pulses. The initial dose of Nanosens was 15 mg/kg.

Optimal regimen for the PLAN procedure with the drug Nanosens: intravenous administration of the drug Nanosens (15 mg/kg or 7 mg/kg), 2–5 min pulsed irradiation (energy density per pulse: 0.6 J/cm2; number of pulses: 100 or 50).

Research was conducted on mice with the following implanted tumors: B16 melanoma, Lewis lung carcinoma LLC, S-37 sarcoma, C-26 carcinoma, Ehrlich tumor, cervical cancer RShM5, and the solid form of leukemia P-388. For experiments, tumors were inoculated into mice under the skin on the outer surface of the thigh in different amounts of tumor tissue per animal. The day of tumor inoculation was considered day zero of its growth. Treatment of mice with P-388, S-37, OE, S-26, and LLC was carried out on days 6–7 after inoculation of tumor material, and mice with CC on days 10–12.

After the tumors had taken root, mice were injected intravenously with the drug Nanosens, and after 2–5 minutes, nanoparticles of zinc phthalocyanine entered the bloodstream and accumulated in the tumors. The tumor was irradiated with a 7 ns pulsed laser with a wavelength of 664 nm, about 100 MW in pulse, while the nanoparticles were heated to temperatures above 400 degrees Celsius and exploded. Fragments of nanoparticles damaged tissues and the circulatory system of the tumor.

When exposed to a pulsed laser on nanoparticles of zinc phthalocyanine in a tumor, in addition to fragments of nanoparticles, a molecular form of zinc phthalocyanine is also formed, which has photosensitizer properties and is suitable for classical photodynamic therapy. In this case, we can talk about targeted delivery of the photosensitizer exactly to the tumor. In this way, it is possible to further enhance the effect of the drug on the tumor, which causes its degradation and death.

Study of the “acute” toxicity of the drug Nanosens. The doses and route of administration of the Nanosens drug to study its “acute” toxicity were chosen based on a single therapeutic dose (TD) of the drug. When studying the “acute” toxicity (tolerability) of Nanosens with the PLAN procedure in mice, the following dose levels were studied: TD; 2.5TD; 5TD; 7.5TD; 10TD. The drug was administered to mice (males and females) once (or in small doses with an interval between injections of no more than 15 min) intravenously in the doses indicated above. Control animals were injected with an isotonic (0.9%) sodium chloride solution. Pulsed irradiation of the hind limb and thigh was performed. 2–5 min after drug administration.

When choosing doses of the drug to study its “chronic” toxicity in rats, we proceeded from the equitherapeutic dose for rats (ETDr), which was calculated on the basis of the maximum therapeutic dose for mice equal to 15 mg/kg, considering the coefficient of interspecies according to the Freireich method (Freireich et al. 1966) and amounted to 7 mg/kg. When studying the “chronic” toxicity of the Nanosens drug in rats, the following dose levels were studied: ETDr; 10ETDr; 30ETDr. Control animals were injected with an isotonic (0.9%) sodium chloride solution. Pulsed irradiation of the hind limb and thigh was performed. 2–5 min after drug administration. Nanosens was administered to rats once a day for 14 days intravenously at the doses indicated above. Control animals were injected with an isotonic (0.9%) sodium chloride solution.

Immunotoxicity study: In the experiments, we used conventional animals to study the effect of the studied drug on delayed-type hypersensitivity (DTH) in linear mice as well as on the humoral immune response in linear mice (hemagglutination reaction). The total number of animals in the study was 60 mice. In the study, we used animals of the same body weight as possible.

Studying pharmacokinetics was carried out using the Spectrometer Ocean Optics (the device was used to measure and record electronic absorption spectra of blood plasma samples after administration of the Nanosens drug) and the Autosizer 2C device (the device was used to measure the size of nanoparticles in blood plasma samples after administration of the Nanosens drug).

While the biodistribution study was carried out using a high-energy pulse laser, a pulsed laser was used to obtain the molecular form of ZnPc from nanoparticles (Nanogsens preparation) through high-energy pulsed irradiation. The study used a pulsed dye laser (emission wavelength 670 nm, pulse energy 45 mJ, pulse duration 8 ns) pumped by the second harmonic of the Nd:Yag laser. In addition, an installation for recording fluorescence spectra was installed (an Ocean Optics optical fiber spectrometer was used to record the fluorescence spectra of the molecular form of ZnPc.).

Tests for effectiveness, chronic toxicity, acute toxicity, and immunotoxicity were carried out. The range of therapeutic doses of the Nanosens drug and the optimal parameters of pulsed laser radiation were determined.

In all cases, after a single PLAN procedure, we observed inhibition of tumor growth, an increase in life expectancy in the case of S-37 sarcoma and Lewis LLC lung carcinoma, and even a complete cure for some animals. Effect leading to inhibition of the growth of the number of transplantable tumors in mice by 100%–50%; an increase in the average life expectancy of animals by 25%–77%; as well as complete cure of the tumor in 8%–25% of mice.

Thus, by summarizing the data obtained during the study of the antitumor effectiveness of the Nanosens using the PLAN method in mice with transplantable tumors of various histogenesis (C-26 carcinoma, Lewis lung carcinoma, Ehrlich carcinoma, RSM5 cervical cancer, P-388 lymphocytic leukemia, S sarcoma-37, and melanoma B-16), it was established that the treatment procedure in the experiment is a highly effective method of antitumor therapy.

It has been established that C-26 carcinoma, S-37 sarcoma, and Lewis lung carcinoma are the most sensitive tumors to this type of treatment; cervical cancer RSM-5, Ehrlich carcinoma, and melanoma B-16 have average sensitivity; and the solid form of lymphocytic leukemia P-388 turned out to be a tumor resistant to treatment with Nanosens using the PLAN method.

Data on the toxicity (Tables 1, 2) of the drug in therapeutic doses were obtained. At the preliminary stage of research, the limit of the semi-lethal dose LD50 of zinc phthalocyanine when administered orally was determined (LD50>10g/kg). It was not possible to establish it more precisely since no deaths of animals were observed. As expected, when testing the drug at therapeutic doses (1 TD=7mg/kg) in the range of 1 to 10 TD for acute and in the range of 1 to 30 TD for chronic toxicity, no death signs of intoxication were observed in animals, although at the maximum dose, individual organs and tissues acquired a blue color.

The immunochemical study shows the results of assessing the effect of the drug Nanosens on the severity of IHRб, which are presented in Tables 3, 4.

According to the data obtained in a model of immune swelling of the paw in mice when stimulated with sheep erythrocytes, it was found that under the influence of a 14-day course of intravenous administration of the study drug at a dose of 10 T (10 times higher than the therapeutic dose for a mouse), the reaction index was significantly lower by 20% than the control indicator, that is, the effect of drugs at a dose of 67.0 mg/kg led to inhibition of the development of immune inflammation.

The results of a study assessing the effect of the Nanosens drug on the humoral immune response of mice are presented in Table 4.

From the data present in Table 4, it can be seen that the determination of the drug Nanosens in experimental doses led to a change in the serum titer, while intravenous administration of the drug at a dose of 67.0 mg/kg caused a significant decrease in their titer of hemagglutinins compared to control values.

When studying biodistribution (Table. 5), it was found that after a single intravenous administration of the drug Nanosens at a therapeutic dose of 7 mg/kg, nanoparticles predominantly accumulate in the liver, lungs, spleen, and kidneys and are then slowly excreted from these organs (for more than 60 days). The peak accumulation of zinc phthalocyanine nanoparticles in tumor tissue was recorded 24 hours after intravenous administration of the drug.

Table 6 presents the pharmacokinetics results. It can be seen that the particle sizes practically do not change during their circulation in the bloodstream for 24 hours.

After intravenous administration of Nanosens at a therapeutic dose of 15 mg/kg, nanoparticles are completely eliminated from the blood of animals within 24 hours. The experimentally determined half-life of nanoparticles in the blood was 1 hour. After intravenous administration of Nanosens, during the entire time of its circulation in the blood (24 hours), there is no significant change in the size of ZnPc nanoparticles.

It was found that Nanosens does not affect blood clotting and does not have hemolytic activity. It was also found that at concentrations of 2 mg/mL and lower, the drug does not have an irritating effect when administered intravenously. The PLAN procedure with the drug Nanosens, which was used in doses ranging from 15 mg/kg to 75 mg/kg, was satisfactorily tolerated by all animals. There was no death of animals from toxicity. No pronounced external signs of intoxication were observed. During a postmortem examination of animals euthanized 90 days after administration of the drug, no pathological changes were detected in the internal organs and tissues of the animals.

The study did not reveal any allergic effects of Nanosens detected in the “Conjunctival test” in pre-sensitized mice at doses of 8 mg/kg and 80 mg/kg.

The essence of the PLAN method is that powerful-pulsed laser radiation is directed at the nanoparticles of the drug introduced into the tumor. As a result, the nanoparticles are locally heated, leading to their microexplosive ablation, which damages the tumor tissue as well as its feeding capillaries. This leads to the suppression of the tumor, halting and inhibiting its growth, and, in some cases, regression and scarring. Thus, PLAN is a non-surgical method for treating superficial tumors. Nanosense can also be used for treatment using the PLAN method combined with photodynamic therapy. In this case, the treatments are administered sequentially.

A new technology has been developed for producing drugs; a photosensor for intravenous administration can be used in the treatment of varicose oncological disease of the skin and mucous membranes using one of the variants of the photodynamic therapy methods, the method of pulse laser ablation of nanoparticles (PLANB). The drug is administered intravenously, and nanoparticles of the drug enter the capillary network of the tumor, where they explode when irradiated with a powerful pulsed laser.

The tumor is irradiated with a powerful pulsed laser with a wavelength in the absorption region of zinc phthalocyanine nanoparticles. Nanoparticles that absorb light at the laser radiation length quickly heat up and explode (nanoparticle ablation), thereby destroying tumor tissues and their capillary systems, which leads to tumor suppression, degradation, and, in some cases, death after a single application.