**3. Influence of the structure of hydrogels hydrogel implants on dynamics of deposition and diffusion of doxorubicin**

It should be noted that it is important to prevent the recurrence of the malignancy after its removal, so it is important that the implant materials contain antitumor drugs.

The hybrid hydrogels used are spatially crosslinked hydrophilic polymers that are characterized by a unique combination of properties such as high hydrophilicity, softness, flexibility and strength, as well as unique biocompatibility [9]. Due to their ability to absorb significant amounts of water and biological fluids, porosity and elasticity, they more than any other synthetic biomaterials resemble human tissues and have been successfully used for decades as a means of targeted transport and prolonged drug release [10], biosensors [11], anti-burn and hemostatic dressings [12], materials for tissue engineering and plastic surgery [13], etc. Given the possibility of using the proposed hybrid hydrogels in the creation of implants that will have the ability to deposit drugs, primarily antimicrobial and antitumor, we thought it appropriate to study the diffusion properties of hybrid hydrogels with different porosity and with immobilized drugs, in particular drugs.

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

**Materials and methods**. The research was conducted in the department of functional hydrogels of the Ovcharenko Institute of Biocolloid Chemistry, NAS of

*Reagents for the synthesis of hydrogels:* Polyvinyl alcohol (PVA) (AppliChem GmbH, 98%; 72 kDa); formaldehyde (LAB-SCAN, 37%); concentrated sulfuric acid (AAc); Triton X-100 (AppliChem GmbH); acrylic acid (99%, Sinbias); ammonium persulfate (PSA) (Thermo Fisher, 98%); N, N′-methylenebisacrylamide

*Medicinal product:* Doxorubicin "Ebeve" - concentrate for solution for infusion containing 2 mg/ml For saturation with pharmaceuticals, hydrogels of different densities were obtained, varying the mass fraction of PVA. The content of PVA in the liquid-crosslinked gel was 7.1 wt. %, and in a tightly crosslinked gel - 8.0%. For the manufacture of a hybrid hydrogel based on pre-synthesized polyvinylformal and acrylic acid, 0.6 g of PVF was placed in a medical syringe with a capacity of 10 ml and impregnated with a solution containing 0.6 ml of AAc, 0.2 ml of 3% MBA solution and 5.25 ml of 40% solution of PSA. After impregnation, 4.5 ml of liquid was squeezed out and the resulting polymerization composition was placed

*Spectral analysis* (FTIR) of wet samples was performed using a spectrometer Spectrum BX FT-IR (Perkin Elmer). The spectra were recorded using the method of disturbed total internal reflection (internal reflection spectroscopy) in the spectral range 4000–550 cm−1 with a resolution of 2 cm−1. Each spectrum was recorded 8

*The porosity* of the samples was determined by gravimetric method according to

1 100% ρ

*<sup>V</sup>* (1)

is the density of the material used [14]. When measur-

 =− × <sup>⋅</sup> *<sup>m</sup> <sup>P</sup>*

where *P* is the total porosity [%], m is the dry weight of the sample, *V* is the

ing the pore size, an optical microscope SIGETA MB 140 LED Mono was used to study the drugs in transmitted, reflected and mixed light. Toup View 3.5 was used to

Detailed information on the structure of the pore space of polymer systems was obtained from the analysis of microphotographs taken using a scanning electron microscope JSM-6060 LA (JEOL, Japan) with a resolution of 4 nm. The polymer samples were freeze-dried in a sublimation unit UZV-10 (Kharkov, Ukraine), attached to standard holders with a double-sided conductive film and covered with a layer of Au/Pd with a thickness of 25 nm in the ion-spray unit Gatan 682 Precision

*The kinetics of swelling* of the samples of the proposed hydrogels was studied at a temperature of 25°С in distilled water and saline (0.9% aqueous sodium chloride solution), determining the degree of swelling of Q samples weighing 23.8–27.0 mg by gravimetric method according to the formula: Qt = (mt – md)/md, where Qt and mt are the degree of swelling and the mass of the swollen sample in a certain time

*Diffusion of doxorubicin in hybrid hydrogels was studied as follows*. Samples of dry hydrogels in the form of cylinders with a diameter of 12 mm and a weight of 50 mg (height varied from 5 mm to 8 mm depending on the composition of the hydrogel) for saturation were placed in 0.02% doxorubicin solution for

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

Ukraine using the following substances and materials:

(Merck) was used without further purification.

in an oven at 40° C for 1 hour.

volume of the dry sample,

ρ

Patching and Coating System Gatan, USA).

process statistical data and video images of the microscope.

interval, md is the initial mass of the dry sample [15, 16].

the formula:

times to prevent accidental artifacts.

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

**Materials and methods**. The research was conducted in the department of functional hydrogels of the Ovcharenko Institute of Biocolloid Chemistry, NAS of Ukraine using the following substances and materials:

*Reagents for the synthesis of hydrogels:* Polyvinyl alcohol (PVA) (AppliChem GmbH, 98%; 72 kDa); formaldehyde (LAB-SCAN, 37%); concentrated sulfuric acid (AAc); Triton X-100 (AppliChem GmbH); acrylic acid (99%, Sinbias); ammonium persulfate (PSA) (Thermo Fisher, 98%); N, N′-methylenebisacrylamide (Merck) was used without further purification.

*Medicinal product:* Doxorubicin "Ebeve" - concentrate for solution for infusion containing 2 mg/ml For saturation with pharmaceuticals, hydrogels of different densities were obtained, varying the mass fraction of PVA. The content of PVA in the liquid-crosslinked gel was 7.1 wt. %, and in a tightly crosslinked gel - 8.0%. For the manufacture of a hybrid hydrogel based on pre-synthesized polyvinylformal and acrylic acid, 0.6 g of PVF was placed in a medical syringe with a capacity of 10 ml and impregnated with a solution containing 0.6 ml of AAc, 0.2 ml of 3% MBA solution and 5.25 ml of 40% solution of PSA. After impregnation, 4.5 ml of liquid was squeezed out and the resulting polymerization composition was placed in an oven at 40° C for 1 hour.

*Spectral analysis* (FTIR) of wet samples was performed using a spectrometer Spectrum BX FT-IR (Perkin Elmer). The spectra were recorded using the method of disturbed total internal reflection (internal reflection spectroscopy) in the spectral range 4000–550 cm−1 with a resolution of 2 cm−1. Each spectrum was recorded 8 times to prevent accidental artifacts.

*The porosity* of the samples was determined by gravimetric method according to the formula:

$$P = \left(1 - \frac{m}{V \cdot \rho}\right) \times 100\% \tag{1}$$

where *P* is the total porosity [%], m is the dry weight of the sample, *V* is the volume of the dry sample, ρis the density of the material used [14]. When measur-

ing the pore size, an optical microscope SIGETA MB 140 LED Mono was used to study the drugs in transmitted, reflected and mixed light. Toup View 3.5 was used to process statistical data and video images of the microscope.

Detailed information on the structure of the pore space of polymer systems was obtained from the analysis of microphotographs taken using a scanning electron microscope JSM-6060 LA (JEOL, Japan) with a resolution of 4 nm. The polymer samples were freeze-dried in a sublimation unit UZV-10 (Kharkov, Ukraine), attached to standard holders with a double-sided conductive film and covered with a layer of Au/Pd with a thickness of 25 nm in the ion-spray unit Gatan 682 Precision Patching and Coating System Gatan, USA).

*The kinetics of swelling* of the samples of the proposed hydrogels was studied at a temperature of 25°С in distilled water and saline (0.9% aqueous sodium chloride solution), determining the degree of swelling of Q samples weighing 23.8–27.0 mg by gravimetric method according to the formula: Qt = (mt – md)/md, where Qt and mt are the degree of swelling and the mass of the swollen sample in a certain time interval, md is the initial mass of the dry sample [15, 16].

*Diffusion of doxorubicin in hybrid hydrogels was studied as follows*. Samples of dry hydrogels in the form of cylinders with a diameter of 12 mm and a weight of 50 mg (height varied from 5 mm to 8 mm depending on the composition of the hydrogel) for saturation were placed in 0.02% doxorubicin solution for

18 hours at a temperature of 25°С. After saturation, the mass of the drug was calculated taking into account changes in the initial concentration. Excess fluid was squeezed from the swollen samples using a disposable medical syringe, the squeezed samples were weighed and placed in vials of 20 ml of saline. Diffusion of doxorubicin was examined by UV spectroscopy using a spectrophotometer-fluorimeter DS-11 FX + (DeNovix, USA), analyzing the samples at certain intervals during the day at a temperature of 25°С and periodic stirring. The concentration of active substances was determined by the normalized peak absorption of doxorubicin at 480 nm.

**Results and discussion.** IR spectroscopy. Based on the obtained IR spectra, the functional groups of porous matrices based on PVF were characterized (**Figure 4**). Wide and intense peaks in the region of about 3362–3382 cm−1 can be attributed to the valence vibrations of hydroxyl groups. The expansion of these absorption bands is explained by the hydrogen bonds that the OH groups join. Bands at 1007 cm−1 on the spectrum of the matrix based on PVF are also characteristic of the valence vibrations of the hydroxyl group of primary alcohols C OH.

According to the calculations performed by formula (1), the high-density crosslinked hydrogel had a porosity of 91.8%, and the low-density crosslinked hydrogel - 95.0%. The calculated porosity of the hydrogel composition with acrylic acid was 85.9%.

According to light-optical and electron microscopy, the obtained hydrogels had a heterogeneous multilevel porous architecture. That is, the pores of the highest level were also formed from porous structures that were about two rows smaller. According to the calculations, the pores of the highest level had a diameter of 120–180 μm in a high-density crosslinked hydrogel (**Figure 5a**) and 460–670 μm in a low-density crosslinked hydrogel (**Figure 5b**). The pore size in the hybrid hydrogel with acrylic acid varied from 200 μm (**Figure 6a**) to 590 μm [15].

For small pores of the lowest level, which form the substructure of the walls of large pores (**Figure 6a**), regardless of the density of the hydrogel, their diameter was 3–5 μm (**Figure 6b**).

The porosity and pore size of the implant material play a significant role in tissue regeneration, so these parameters are widely studied and discussed in numerous studies. The porous structure of the matrix is necessary for tissue regeneration, because it depends on the adhesion, migration and proliferation of cells, as well as the diffusion of nutrients, oxygen and metabolites. It has been found that large pores provide nutrient delivery and removal of metabolic products, while small pores provide a larger surface area for cell adhesion [10, 17]. It should be noted that the influence of pore architecture on the behavior of cells also depends on

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newly formed vessels [21].

*b - pores of the substructural level.*

hydrogel samples with drugs.

ensure regeneration processes in biological tissues.

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

*A and b. porous structure of high-density crosslinked (a) and low-density cross-linked (b) PVF hydrogels* 

their nature. *In vitro* experiments have shown that 380–405 μm pores are best for chondrocytes and osteoblasts, while fibroblasts are prone to proliferation in smaller diameter pores (186–200 μm) [18]. Such preferences may be explained by the fact that, although large pores improve the diffusion of nutrients and oxygen, fibroblasts tend to cling to the substrate with smaller pores, as this increases the area of specific contact [17]. According to in vitro studies, greater porosity is accompanied by increased cell migration and infiltration [19, 20]. At the same time, in vivo it can be the cause of protein leaching [17, 21]. Also an important characteristic is the interporous connection, which depends not only on the diffusion properties of the matrix in relation to nutrients and oxygen, but also the possibility of ingrowth of

*A and b. electron microscopic image of the pore architecture of PVF hydrogels: A - pores of the highest level,* 

Based on this, it can be argued that our proposed hydrogels are able to accumulate and transport through a system of small pores of various metabolites and drugs, as well as serve as a matrix for attachment and migration of different cell types that

Kinetics of hydrogel swelling. From the analysis of the kinetics of swelling of hydrogels of different structure it can be concluded that all samples of hydrogels in water reach an equilibrium state of swelling in the first 30 minutes. In saline, the degree of swelling of the hydrogels was slightly lower (approximately 16%), but this had little effect on the high rate of swelling. Only in the composite hydrogel with acrylic acid, the equilibrium state in saline was reached within 3 hours. This pattern is inherent in hydrogels in general and is explained by a decrease in ionic osmotic pressure, which causes swelling of hydrogels, with increasing ionic strength of the solution. The obtained results were used to determine the period of saturation of

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

*according to the processing of microscopic images 40x.*

**Figure 5.**

**Figure 6.**

**Figure 4.** *IR spectrum of a porous matrix based on PVF.*

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

#### **Figure 5.**

*A and b. porous structure of high-density crosslinked (a) and low-density cross-linked (b) PVF hydrogels according to the processing of microscopic images 40x.*

#### **Figure 6.**

*A and b. electron microscopic image of the pore architecture of PVF hydrogels: A - pores of the highest level, b - pores of the substructural level.*

their nature. *In vitro* experiments have shown that 380–405 μm pores are best for chondrocytes and osteoblasts, while fibroblasts are prone to proliferation in smaller diameter pores (186–200 μm) [18]. Such preferences may be explained by the fact that, although large pores improve the diffusion of nutrients and oxygen, fibroblasts tend to cling to the substrate with smaller pores, as this increases the area of specific contact [17]. According to in vitro studies, greater porosity is accompanied by increased cell migration and infiltration [19, 20]. At the same time, in vivo it can be the cause of protein leaching [17, 21]. Also an important characteristic is the interporous connection, which depends not only on the diffusion properties of the matrix in relation to nutrients and oxygen, but also the possibility of ingrowth of newly formed vessels [21].

Based on this, it can be argued that our proposed hydrogels are able to accumulate and transport through a system of small pores of various metabolites and drugs, as well as serve as a matrix for attachment and migration of different cell types that ensure regeneration processes in biological tissues.

Kinetics of hydrogel swelling. From the analysis of the kinetics of swelling of hydrogels of different structure it can be concluded that all samples of hydrogels in water reach an equilibrium state of swelling in the first 30 minutes. In saline, the degree of swelling of the hydrogels was slightly lower (approximately 16%), but this had little effect on the high rate of swelling. Only in the composite hydrogel with acrylic acid, the equilibrium state in saline was reached within 3 hours. This pattern is inherent in hydrogels in general and is explained by a decrease in ionic osmotic pressure, which causes swelling of hydrogels, with increasing ionic strength of the solution. The obtained results were used to determine the period of saturation of hydrogel samples with drugs.

#### **3.1 Diffusion of doxorubicin from hydrogels**

The diffusion kinetics of doxorubicin are shown in **Figure 7**. The low-density cross-linked hydrogel sorbs twice as much doxorubicin as the high-density crosslinked hydrogel, possibly due to the absence of steric obstacles to penetration of the porous hydrogel structure by a large drug molecule (molecular weight is 544 g/mol). In *in vitro* experiments, the former hydrogel provided a 3–4-fold greater drug concentration in the environment compared to the latter hydrogel (**Figure 7**). The latter hydrogel, however, allows for a smoother and more prolonged drug release profile and therefore it is advisable to use for implants with a prolonged drug effect, while low-density crosslinked hydrogel - for urgent release of a shock dose of cytostatic preparation.

Hybrid hydrogels based on PVF and incorporated poly-AAc have a much greater about an order of magnitude - the ability to deposit doxorubicin (compared to PVF). This effect may be due to the formation of ionic bonds between the active -COOH groups present in the hybrid hydrogel and the amine groups present in the structure of doxorubicin. The slow hydrolysis of these ionic bonds explains the prolonged (several days) release of doxorubicin from hybrid hydrogels (**Figure 8**).

This prolonged ability of hybrid hydrogel implants will facilitate their use for the deposition of antitumor drugs and maintain their effective concentration in the pathological focus.

Thus, studies have shown that the kinetics of diffusion of drugs from liquidcrosslinked hydrogel reaches a minimum therapeutic level within a few minutes, whereas in the case of densely crosslinked hydrogel diffusion begins with a delay of

**Figure 7.**

*Diffusion of doxorubicin from low-density crosslinked (1, 2) and high-density cross-linked (3, 4) hydrogel during the day (\_\_\_\_\_ - release into solution;* −*----- -release percentage).*

**Figure 8.**

*Diffusion of doxorubicin from saturated samples of composite hydrogel containing polyacrylic acid during the week; A-concentration,* μ*g/ml; B-percent release from sorbed.*

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

several hours and the amount of drug released at equilibrium is much lower. Values (20–25%). It has also been found 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

It is important to note that the main factor in antitumor therapy is the temporary parameter of tissue saturation with drugs, in this regard, we continue to study in this direction. However, this direction of antitumor therapy is accompanied by

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

Noteworthy is the delivery of doxorubicin to tumor cells through the use of

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%

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

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

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.

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

[24]. This necessitates the development of new therapeutic systems for

developed congestive heart failure,

, and 36% with cumulative dosages higher than

polymer, and also provides a smoother, prolonged release of the drug.

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

of patients receiving dosages of 500–550 mg/m2

actively targeting tumor cells through endocytosis.

transport of anticancer drugs, primarily doxorubicin.

18% with dosages of 551–600 mg/m2

surgery.

nanomaterials [22].

601 mg/m2

promising.

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

several hours and the amount of drug released at equilibrium is much lower. Values (20–25%). It has also been found 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.

It is important to note that the main factor in antitumor therapy is the temporary parameter of tissue saturation with drugs, in this regard, we continue to study in this direction. However, this direction of antitumor therapy is accompanied by surgery.
