**4. Nanoparticles, possible way of delivery of doxorubicin to tumor cells**

Noteworthy is the delivery of doxorubicin to tumor cells through the use of nanomaterials [22].

In recent years, researchers have focused on the so-called "smart" therapeutic systems that are able to respond to minor changes in their environment by a sharp change in their physicochemical (primarily diffusion) characteristics [23]. The greatest attention is paid to thermo- and pH-sensitive hydrogels, which under the influence of minor, physiologically acceptable changes in temperature or pH are capable of controlled mass release of drugs, in particular, anticancer [21].

Even, doxorubicin is one of the most effective and widely used drugs against a wide range of cancers. But, its clinical use in parenteral administration is accompanied by such side effects as high cardiac toxicity and myelosuppression. The most serious long-term adverse effect of doxorubicin therapy is irreversible cardiomyopathy, which is based on the total cumulative dose [22]. In one clinical study, ~4% of patients receiving dosages of 500–550 mg/m2 developed congestive heart failure, 18% with dosages of 551–600 mg/m2 , and 36% with cumulative dosages higher than 601 mg/m2 [24]. This necessitates the development of new therapeutic systems for the transport of doxorubicin, increasing its therapeutic efficacy and minimizing side effects. Ideally, therapeutic transport systems of doxorubicin should inhibit the release of the drug in plasma and release it only after reaching the tumor site by actively targeting tumor cells through endocytosis.

As a trigger for targeting the therapeutic transport system of doxorubicin can be used a significant difference in the pH of plasma (pH = 7.4) and the microenvironment of the tumor (pH = 6.5) and lysosomes (pH = 4.8–5) [6, 9, 21]. In particular, this determines the prospects for the use of pH-sensitive hydrogels for controlled transport of anticancer drugs, primarily doxorubicin.

However, nanogels that are sensitive to changes in both pH and temperature, such as, for example, hydrogel copolymers synthesized by us based on N-isopropylacrylamide (NIPAm) and acrylic acid (AAc), seem to be especially promising.

The synthesis of nanogels based on NIPA was described in detail earlier [25, 26]. N-isopropylacrylamide, NIPAm (Sigma-Aldrich, 97%) was recrystallized from hexane and dried under vacuum; N,N′-methylenebisacrylamide (MBA) (Merck,98%), acrylic acid (AAc) was purified by distillation and subsequent fractional distillation, potassium persulphate, PSP (Sigma 98%)were used without additional purification, as well as sodium dodecylsulphate (SDS), polyethylenimine (Sigma-Aldrich ММ 2000 Da) and iron salts (FeSO4 and FeCl3) used in magnetite synthesis. Briefly, 2,3 g of NiPAm, 0,0393 g of MBA, 0,1124 g of SDS, 5 mL of magnetite suspension, 0,115 g of AAc and 135 g of water were placed in the beaker.

After that the beaker was set on a magnetic stirrer to dissolve the reagents at room temperature. At the end of the mixing the solution in the beaker underwent purification with argon for 2 minutes. It was then transferred into the glass reactor equipped with a stirrer and thermometer. The reactor was placed in a water bath with that was maintained at constant temperature. The synthesis was carried out at 68–70°C. When the reactor temperature reaches these temperature, 10 mL of 0,93% solution of PSP in water was added. The mixing rate was 500 rotations per minute. The duration of the synthesis took additional 6 hours. The release kinetics of the antitumor drug doxorubicin were investigated using a UV spectrometer "Specord M 40" (maximum absorption 480 nm).

The size of the polymer carriers (transport systems) of anticancer drugs is of great importance, since nanoparticles with a diameter of less than 200 nm, on the one hand, are able to penetrate into cells, in particular, affected cells, and on the other hand, they are not captured by macrophages, which contributes to an increase in the duration of their circulation in the body. As can be seen from the Electronic Microphotographs (TEMs) shown in **Figure 9**, the synthesized nanogels are characterized by uniformity of shape and size and have an average diameter of about 100 nm. At magnification (see **Figure 9**, box), you can see incorporated into the nanopherogel nanoparticles of magnetite with a size of about 10 nm.

The obtained images correlate well with the results of dynamic light scattering measurements. It is shown that the average size of the synthesized hydrogels based on NIPAm and AAc is about 100–200 nm and depends on the temperature and pH value, as well as the value of the zeta potential of nanoparticles. Synthesized copolymer nanogels based on NIPAm and AAc combine thermo- and pH-sensitivity. When heated above the LCST (lower critical solution temperature, equal for NIPAm to a temperature of 32–34°C) and when the environment is acidified, the diameter of the nanoparticles decreases. Thus, it was found that the average size of nanoparticles of copolymer hydrogels based on NIPAA and AAc when heated from 25 to 50°С decreases by 3–5 times, which is a consequence of thermo-induced phase transition from swollen to collapsed state of the hydrogel (**Figure 10a** and **b**).

At the same time, in the acidic region, the pH of nanopherogels is about 10 nm, while in an alkaline environment increases by about an order of magnitude. Note that these processes are reversible and further cooling of the nanogels (as well as an

#### **Figure 9.**

*Microphotographs (TEM) of synthesized nano(ferro)gels based on NIPAm - AAc copolymer with incorporated magnetite.*

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of the system.

**Figure 10.**

**Figure 11.**

*and pH = 12.0 (2).*

*Improving the Antitumor Effect of Doxorubicin in the Treatment of Eyeball and Orbital Tumors*

*A and b. size distribution of nano(ferro)gels based on NIPAm – AAc copolymer with incorporated magnetite depending on: A - temperature at 50°*С *(1), 37°*С *(2) and 25°*С *(3); b - acidity of the medium at pH = 1.1 (1)* 

increase in pH) leads to an increase in their size to the original values. This behavior of nanosized hydrogel matrices creates the preconditions for spontaneous targeted release of incorporated antitumor drugs, primarily doxorubicin when heated in a temperature-acceptable range, for example, in drug hyperthermia or in contact

*A and b. size distribution (a) and zeta potential (b) of nano(ferro)gels based on NIPAm- AAc copolymer with* 

*incorporated magnetite and doxorubicin at temperatures of 25°*С *(1) 37°*С *(2) and 50°*С *(3).*

Zeta potential as a function of the surface charge of a substance in a liquid is an excellent characteristic of electrostatic repulsion between particles. Zeta potential is usually used to predict and control the stability of the dispersion. Moreover, the characteristics of the solid–liquid interface can have a strong effect, in particular on adhesion, flotation, and in more concentrated systems on the rheological behavior

Thus, it shown that an increase in temperature in the range of 25°С to 37°С, up to 50°С leads to an increase (in absolute value) of the zeta potential and a decrease in the size of ferrogel nanoparticles, which indicates an increase in aggregate stability of the corresponding colloidal systems. This pattern can be explained by the rupture of intermolecular hydrogen bonds that promote aggregation, with increas-

Thermo- and pH-sensitive copolymer hydrogels based on NIPAm and AAc with incorporated magnetite and cytostatic doxorubicin were also characterized using a Zetasizer Nano ZS (Malvern Instruments) zeta-seiser. It was demonstrated (**Figure 11a** and **b**) that as the temperature increases (in the physiologically acceptable range), the size of nanoparticles decreases (to about 50 nm) and

with affected cells, which are characterized by acidic pH.

ing Brownian motion when heating NIPAm macromolecules.

*DOI: http://dx.doi.org/10.5772/intechopen.95080*

*Improving the Antitumor Effect of Doxorubicin in the Treatment of Eyeball and Orbital Tumors DOI: http://dx.doi.org/10.5772/intechopen.95080*

**Figure 10.**

*A and b. size distribution of nano(ferro)gels based on NIPAm – AAc copolymer with incorporated magnetite depending on: A - temperature at 50°*С *(1), 37°*С *(2) and 25°*С *(3); b - acidity of the medium at pH = 1.1 (1) and pH = 12.0 (2).*

**Figure 11.**

*A and b. size distribution (a) and zeta potential (b) of nano(ferro)gels based on NIPAm- AAc copolymer with incorporated magnetite and doxorubicin at temperatures of 25°*С *(1) 37°*С *(2) and 50°*С *(3).*

increase in pH) leads to an increase in their size to the original values. This behavior of nanosized hydrogel matrices creates the preconditions for spontaneous targeted release of incorporated antitumor drugs, primarily doxorubicin when heated in a temperature-acceptable range, for example, in drug hyperthermia or in contact with affected cells, which are characterized by acidic pH.

Zeta potential as a function of the surface charge of a substance in a liquid is an excellent characteristic of electrostatic repulsion between particles. Zeta potential is usually used to predict and control the stability of the dispersion. Moreover, the characteristics of the solid–liquid interface can have a strong effect, in particular on adhesion, flotation, and in more concentrated systems on the rheological behavior of the system.

Thus, it shown that an increase in temperature in the range of 25°С to 37°С, up to 50°С leads to an increase (in absolute value) of the zeta potential and a decrease in the size of ferrogel nanoparticles, which indicates an increase in aggregate stability of the corresponding colloidal systems. This pattern can be explained by the rupture of intermolecular hydrogen bonds that promote aggregation, with increasing Brownian motion when heating NIPAm macromolecules.

Thermo- and pH-sensitive copolymer hydrogels based on NIPAm and AAc with incorporated magnetite and cytostatic doxorubicin were also characterized using a Zetasizer Nano ZS (Malvern Instruments) zeta-seiser. It was demonstrated (**Figure 11a** and **b**) that as the temperature increases (in the physiologically acceptable range), the size of nanoparticles decreases (to about 50 nm) and the zeta potential increases, which indicates an increase in the aggregate stability of nanosuspensions.

Taking in the account medical field of application of synthesized nano(ferro) gels, an extremely important problem is their washing from the unreacted monomers and other toxic pristine materials since the gelation reaction never proceeds with 100% conversion. Washing of medical nanogels from monomer residues and unreacted initiators is carried out by long-term extraction (for 4–10 days) with a suitable solvent (preferably water) with its repeated replacement.

As it can be seen from **Figure 12**, the immediately after synthesis, the concentration of NIPAm monomer significantly exceeds the maximum allowable level. After washing 5 times, the concentration of monomer decreases 50 times, and after seven times - more than 500. Analyzing the size distribution spectra of the crude nanogel samples and the corresponding samples after washing, obtained by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments), we can conclude that due to washing using diafiltration the average size of nanoparticles increases slightly, which, in our opinion, is associated with the leaching of surface-active sodium dodecyl sulfate, which prevented the aggregation of nanoparticles [27].

It can be seen from **Figure 12**, the concentration of NIPAm monomer significantly exceeds the maximum allowable level immediately after the synthesis. While, after washing 5 times, the concentration of monomer decreases 50 times, and after seven times – drop down more than 500 times.

In the case of macrogels, the temperature of their phase transition between the swollen and collapsed state (which determines the possibility of controlling their physicochemical properties, primarily diffusion) is determined gravimetrically, which is almost impossible in the case of nanogels. However, it has been demonstrated that the phase transition temperature can be determined no less accurately by measuring light transmission. As can be seen from **Figure 13**, (□), at temperatures below 30°С hydrogels are in the expanded conformation, while when heated above 32°С (lower critical solution temperature, LCST) there is a phase transition to a compact collapsed state due to the destruction of hydrogen bonds between molecules water and hydrophilic amide groups of NIPAm caused by Brownian motion, as well as the strengthening of hydrophobic interactions of isopropyl groups of NIPAm. As a result, there was a sharp decrease in the light transmission of dispersions, and the phase transition temperature for nanopherogels was about 35°С. A small increase in the magnitude of the phase transition from the inherent NIPAm temperature of 32°С is explained by its copolymerization with hydrophilic acrylic acid. Due to the established effect of increasing the NIPA phase transition temperature when copolymerized with a more hydrophilic monomer (eg acrylamide or AAc) and decreasing the NIPAm phase transition temperature when copolymerized with a more hydrophobic monomer (eg acrylonitrile), therapeutic systems based

**Figure 12.**

*A and b. reduction of absorption (a) of monomeric NIPAm in the UV region (1-after washing; 2-immediately after synthesis); change in the concentration of NIPAm depending on the number of washes (b).*

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particular doxorubicin.

**Figure 13.**

**Figure 14.**

*Improving the Antitumor Effect of Doxorubicin in the Treatment of Eyeball and Orbital Tumors*

*The effect of temperature on light transmission for crude (*□*) and purified (◊)nano(ferro)gels based on* 

on delivery and controlled release at the desired temperature of various drugs, in

The analysis of **Figure 14** shows that at 25°С the release of the antitumor drug doxorubicin is completed after 30–40 min, whereas heating above the LCST causes spontaneous release of the drug from the collapsed hydrogel. Thus, the synthesized nano (ferro) gels based on NIPAm, AAc and magnetite due to their unique properties are a promising material for the creation of therapeutic systems of targeted delivery and controlled release of drugs, in particular, in drug hyperthermia.

*Kinetics of release of doxorubicin from nano(ferro)gels based on NIPAm and AAc with incorporated magnetite at pH = 7 (nano(ferro)gel was saturated with a solution of the drug with a concentration of 2.5x10−2%).*

Incorporation of pre-synthesized nanosized magnetite into nanogels allows to give the corresponding nanopherogels magnetically controlled properties, namely - the possibility of targeted localization under the influence of a constant magnetic field of anticancer drug carriers in close proximity to the target organ, which is extremely important. Means and the need to minimize their overall impact on the body. Thus, the incorporation into the composition of hydrogel matrices of nanosized magnetite provides the possibility of targeted localization of the developed therapeutic systems in close proximity to the target organ by applying a constant low-intensity magnetic field with subsequent controlled release of incorporated drugs (primarily, cancer-free - or low-intensity alternating magnetic field.

For nano(ferro)gels on the base of NIPAm, biological studies were performed that showed that magnetic hydrogels with a magnetite content of up to 10% are not toxic to PTP cells (primary swine testicle) (**Figure 15a**). Moreover, upon contact of the original matrix with the cells, it was found that their activity tends to increase compared to the activity of control intact cells. Therefore, the result allows us to

*DOI: http://dx.doi.org/10.5772/intechopen.95080*

*NIPAm and AAc with incorporated magnetite at pH = 7.*

*Improving the Antitumor Effect of Doxorubicin in the Treatment of Eyeball and Orbital Tumors DOI: http://dx.doi.org/10.5772/intechopen.95080*

**Figure 13.**

*The effect of temperature on light transmission for crude (*□*) and purified (◊)nano(ferro)gels based on NIPAm and AAc with incorporated magnetite at pH = 7.*

#### **Figure 14.**

*Kinetics of release of doxorubicin from nano(ferro)gels based on NIPAm and AAc with incorporated magnetite at pH = 7 (nano(ferro)gel was saturated with a solution of the drug with a concentration of 2.5x10−2%).*

on delivery and controlled release at the desired temperature of various drugs, in particular doxorubicin.

The analysis of **Figure 14** shows that at 25°С the release of the antitumor drug doxorubicin is completed after 30–40 min, whereas heating above the LCST causes spontaneous release of the drug from the collapsed hydrogel. Thus, the synthesized nano (ferro) gels based on NIPAm, AAc and magnetite due to their unique properties are a promising material for the creation of therapeutic systems of targeted delivery and controlled release of drugs, in particular, in drug hyperthermia.

Incorporation of pre-synthesized nanosized magnetite into nanogels allows to give the corresponding nanopherogels magnetically controlled properties, namely - the possibility of targeted localization under the influence of a constant magnetic field of anticancer drug carriers in close proximity to the target organ, which is extremely important. Means and the need to minimize their overall impact on the body. Thus, the incorporation into the composition of hydrogel matrices of nanosized magnetite provides the possibility of targeted localization of the developed therapeutic systems in close proximity to the target organ by applying a constant low-intensity magnetic field with subsequent controlled release of incorporated drugs (primarily, cancer-free - or low-intensity alternating magnetic field.

For nano(ferro)gels on the base of NIPAm, biological studies were performed that showed that magnetic hydrogels with a magnetite content of up to 10% are not toxic to PTP cells (primary swine testicle) (**Figure 15a**). Moreover, upon contact of the original matrix with the cells, it was found that their activity tends to increase compared to the activity of control intact cells. Therefore, the result allows us to

**Figure 15.**

*a and b. Cytotoxicity of nano(ferro)gels based on NIPAm for PTP cells (a) and HEP-2 cells (b).*

consider nano(ferro)gels based on NIPAm hydrogels, suitable for the development of hyperthermia of cancer cells, targeted delivery and controlled release of drugs, as well as objects for cell growth. Similar results were obtained in the study of cytotoxicity for HEP-2 cells (epidermal carcinoma of larynx) (**Figure 15b**).

For copolymer ferrogels with a 95% NIPAA content, biological studies performed showed that magnetic hydrogels with a magnetite content of up to 10% are not toxic to PTP cells (**Figure 15a**). Moreover, upon contact of the original matrix with the cells, it was found that their activity tends to increase compared to the activity of control intact cells. Therefore, the result allows us to consider ferrogels based on copolymer hydrogels, which contain 95% NIPAA and 5% AA, suitable for the development of hyperthermia of cancer cells, targeted delivery and controlled release of drugs, as well as objects for cell growth. Similar results were obtained in the study of cytotoxicity for HEP-2 cells (**Figure 15b**).

#### **5. Conclusion**

From the above points, it can be concluded that the hydrogel implant developed by us will allow to fill soft tissue structures quite effectively during tissue resection. However, this will partially solve the problem. The clinician always faces such an important task as to avoid tumor recurrence. Immobilization and diffusion of doxorubicin into the implant showed that the kinetics of diffusion of the drug from the liquid-crosslinked hydrogel reaches a minimum therapeutic level within a few minutes, whereas in the case of densely crosslinked hydrogel diffusion begins with a delay of several hours and the amount is released. Much smaller values (20–25%). It is also shown that the tightly crosslinked hydrogel has a higher ability to deposit doxorubicin, and therefore, it is advisable to use for implants with a prolonged antibacterial effect, while the liquid crosslinked hydrogel - for the immediate release of a shock dose of antiseptic. It is important to note that the liquid-crosslinked hydrogel absorbs twice as much cytostatics as doxorubicin, which may be due to the lack of steric barriers to the penetration of the bulk molecule of doxorubicin (molecular weight 544 g/mol) into its porous structure. This hydrogel provides in vitro experiments 3–4 times higher concentration in the environment compared to densely crosslinked polymer, and also provides a smoother, prolonged release of the drug.

The obtained preliminary experimental results allow us to conclude that our developed pathways for the delivery of drugs, in particular, doxorubicin to tumor cells will increase the effectiveness of antitumor therapy. We are faced with many questions that we will implement in further research.

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**Author details**

Odessa, Ukraine

Anatoliy Parfentievich Maletskyy1

3 Lviv Regional Clinical Hospital, Lviv, Ukraine

provided the original work is properly cited.

\*Address all correspondence to: maletskiy@filatov.com.ua

and Natalia Mikhailivna Bigun3

*Improving the Antitumor Effect of Doxorubicin in the Treatment of Eyeball and Orbital Tumors*

\*, Yuriy Markovich Samchenko2

1 The Filatov Institute of Eye Diseases and Tissue Therapy, NAMS of Ukraine,

2 Ovcharenko Institute of Biocolloid Chemistry, NAS of Ukraine, Kyiv, Ukraine

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*DOI: http://dx.doi.org/10.5772/intechopen.95080*

*Improving the Antitumor Effect of Doxorubicin in the Treatment of Eyeball and Orbital Tumors DOI: http://dx.doi.org/10.5772/intechopen.95080*
