*2.5. The Investigation of the Chitosan-Coated Liposomes Entrapping DEX on the Nociceptive Reactivity in Rats*

The somatic nociceptive reactivity was assessed using the hot plate test (Ugo-Basile apparatus), in order to measure the latency time response to thermal paw noxious stimulation [36]. The noted baseline latency (before the substances administration) was 4.3 ± 0.2 s (mean ± standard deviation of mean, S.D.). The suggested cut-off time of 12 s was considered to avoid damage to the paws. The paws' withdrawal latency (seconds) was counted before the test, and 15, 30, 60, 90, 120 min, and 4, 6, 8, 10, 12 h after administration of the tested substances. Variances between the measured and baseline latencies were estimated as an index of analgesia. The prolongation of the latency time reactivity was suggestive for the analgesic effect, while the diminution in the paws' reaction was indicative of hyperalgesia [37,38].

Moreover, the withdrawal threshold data from paws response measurements were transformed to the percentage maximum possible antinociceptive effect (%MPE) using the following formula [39]:

#### %MPE = [(measured latency-baseline latency)/(cut-off time − baseline latency)] × 100 (1) Committee on Research of 'Grigore T. Popa' University of Medicine and Pharmacy, in

%MPE = [(measured latency-baseline latency)/(cut-off time − baseline latency)] × 100 [39] (1)

*2.5.The Investigation of the Chitosan-Coated Liposomes Entrapping DEX on the Nociceptive* 

The somatic nociceptive reactivity was assessed using the hot plate test (Ugo-Basile apparatus), in order to measure the latency time response to thermal paw noxious stimulation [36]. The noted baseline latency (before the substances administration) was 4.3 ± 0.2 s (mean ± standard deviation of mean, S.D.). The suggested cut-off time of 12 s was considered to avoid damage to the paws. The paws' withdrawal latency (seconds) was counted before the test, and 15, 30, 60, 90, 120 min, and 4, 6, 8, 10, 12 h after administration of the tested substances. Variances between the measured and baseline latencies were estimated as an index of analgesia. The prolongation of the latency time reactivity was suggestive for the analgesic effect, while the diminution in the paws' reaction was

Moreover, the withdrawal threshold data from paws response measurements were transformed to the percentage maximum possible antinociceptive effect (%MPE) using

The protocol of the study was approved (Protocol No. 19157/19.10.2009) by the Ethic

*Polymers* **2021**, *13*, x FOR PEER REVIEW 5 of 14

The protocol of the study was approved (Protocol No. 19157/19.10.2009) by the Ethic Committee on Research of 'Grigore T. Popa' University of Medicine and Pharmacy, in Iasi, Romania, according to the European Ethical Regulations and to the guidelines of IASP Committee for Research and Ethical Issues [40,41]. The data were statistically evaluated using the SPSS 17.0 software for Windows and the ANOVA one-way method. *p*-values less than 0.05 were interpreted as statistically significant compared to the control group. Iasi, Romania, according to the European Ethical Regulations and to the guidelines of IASP Committee for Research and Ethical Issues [40,41]. The data were statistically evaluated using the SPSS 17.0 software for Windows and the ANOVA one-way method. *p*-values less than 0.05 were interpreted as statistically significant compared to the control group. **3. Results** 

#### **3. Results** *3.1. Characterization of Liposomes*

*Reactivity in Rats* 

#### *3.1. Characterization of Liposomes* New carrier systems containing DEX were designed. Microscopic observations of

indicative of hyperalgesia [37,38].

the following formula:

New carrier systems containing DEX were designed. Microscopic observations of liposomes revealed their spherical shape, and the image of nanovesicles containing DEX in optical microscopy is presented in Figure 1. liposomes revealed their spherical shape, and the image of nanovesicles containing DEX in optical microscopy is presented in Figure 1.

**Figure 1.** The optical microscopy image of nano-dexketoprofen (DEX) (sizing scale represents 10 **Figure 1.** The optical microscopy image of nano-dexketoprofen (DEX) (sizing scale represents 10 µm).

μm). The image obtained through optical DIC microscopy presents nano-DEX having regular shape (close to spherical), and well-dispersed due to stabilization with chitosan, prior to dyalization. The size distribution histogram was obtained by measuring the size The image obtained through optical DIC microscopy presents nano-DEX having regular shape (close to spherical), and well-dispersed due to stabilization with chitosan, prior to dyalization. The size distribution histogram was obtained by measuring the size diameter of vesicles from the microscopic image; this suggests formation of vesicles with size 1.4–1.5 µm, in concordance with hydrodynamic size measurements (Figure 1).

The analysis of liposome solutions revealed their mid-range of polydispersity (between 0.435 and 0.636). During the preparation of liposomes, a pH value of 7.12 was noted for DEX encapsulated in liposome solution, and a pH value of 3.84 for the obtained solution after adding chitosan. Because the stabilization of the vesicles with chitosan led to a major decrease in pH value, prolonged dialysis was required to achieve an optimal pH value for gastric absorption; finally, a solution with a pH value of 6.89 was obtained (Table 1).


**Table 1.** Characteristics of liposome solutions.

DEX: dexketoprofen.

profen.

profen.

From the analysis of hydrodynamic size distributions and of the Zeta potentials, a stiffening of the lipid bilayer of the vesicles was noted. Thus, the suspension stability increased, accompanied by an increase in the size of nano-DEX vesicles. Positive charging of the vesicle surface due to protonation created repulsion forces between the vesicles, preventing agglomeration. It can be said that these vesicular systems meet the criteria of colloidal solutions, because a Zeta potential of 61.7 mV for the vesicles containing DEX stabilized with chitosan falls in a very good stability domain. Dialyzation led to a diminution of the vesicle size, and also to a significant decrease in suspension stability up to the agglomeration threshold of 3.89 mV. These phenomena are a consequence of the deprotonation of chitosan amino groups at the surface of the vesicle bilayer in dialysis conditions [42,43] (Figures 2 and 3). creased, accompanied by an increase in the size of nano-DEX vesicles. Positive charging of the vesicle surface due to protonation created repulsion forces between the vesicles, preventing agglomeration. It can be said that these vesicular systems meet the criteria of colloidal solutions, because a Zeta potential of 61.7 mV for the vesicles containing DEX stabilized with chitosan falls in a very good stability domain. Dialyzation led to a diminution of the vesicle size, and also to a significant decrease in suspension stability up to the agglomeration threshold of 3.89 mV. These phenomena are a consequence of the deprotonation of chitosan amino groups at the surface of the vesicle bilayer in dialysis conditions [42,43] (Figures 2and 3). creased, accompanied by an increase in the size of nano-DEX vesicles. Positive charging of the vesicle surface due to protonation created repulsion forces between the vesicles, preventing agglomeration. It can be said that these vesicular systems meet the criteria of colloidal solutions, because a Zeta potential of 61.7 mV for the vesicles containing DEX stabilized with chitosan falls in a very good stability domain. Dialyzation led to a diminution of the vesicle size, and also to a significant decrease in suspension stability up to the agglomeration threshold of 3.89 mV. These phenomena are a consequence of the deprotonation of chitosan amino groups at the surface of the vesicle bilayer in dialysis conditions [42,43] (Figures 2and 3).

*Polymers* **2021**, *13*, x FOR PEER REVIEW 6 of 14

*Polymers* **2021**, *13*, x FOR PEER REVIEW 6 of 14

diameter of vesicles from the microscopic image; this suggests formation of vesicles with

diameter of vesicles from the microscopic image; this suggests formation of vesicles with

The analysis of liposome solutions revealed their mid-range of polydispersity (between 0.435 and 0.636). During the preparation of liposomes, a pH value of 7.12 was noted for DEX encapsulated in liposome solution, and a pH value of 3.84 for the obtained solution after adding chitosan. Because the stabilization of the vesicles with chitosan led to a major decrease in pH value, prolonged dialysis was required to achieve an optimal pH value for gastric absorption; finally, a solution with a pH value of 6.89 was

The analysis of liposome solutions revealed their mid-range of polydispersity (between 0.435 and 0.636). During the preparation of liposomes, a pH value of 7.12 was noted for DEX encapsulated in liposome solution, and a pH value of 3.84 for the obtained solution after adding chitosan. Because the stabilization of the vesicles with chitosan led to a major decrease in pH value, prolonged dialysis was required to achieve an optimal pH value for gastric absorption; finally, a solution with a pH value of 6.89 was

From the analysis of hydrodynamic size distributions and of the Zeta potentials, a

stiffening of the lipid bilayer of the vesicles was noted. Thus, the suspension stability in-

stiffening of the lipid bilayer of the vesicles was noted. Thus, the suspension stability in-

size 1.4–1.5 µm, in concordance with hydrodynamic size measurements (Figure 1).

size 1.4–1.5 µm, in concordance with hydrodynamic size measurements (Figure 1).

**Figure 2.** Size distribution by number of nano-DEX in aqueous solution.nano-DEX: liposomes **Figure 2.** Size distribution by number of nano-DEX in aqueous solution.nano-DEX: liposomes containing dexketoprofen. containing dexketoprofen.

**Figure 3.** Distribution of the nano-DEX Zeta potential. nano-DEX: liposomes containing dexketo-**Figure 3.** Distribution of the nano-DEX Zeta potential. nano-DEX: liposomes containing dexketoprofen.

**Figure 3.** Distribution of the nano-DEX Zeta potential. nano-DEX: liposomes containing dexketo-

**Liposomes Types pH**

Chitosan-coated liposomes

Dialyzed chitosan-coated

#### *3.2. In Vitro Release of DEX from Vesicles Stabilized with Chitosan* Liposomes entrapping DEX 7.12 0.472 368 0.64 Threshold of agglomeration

**Colloidal Solution Characterization**

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**Table 1.** Characteristics of liposome solutions.

**Z-Average Diameter (nm)**

**Polydispersity Index**

wavelength.

somes containing dexketoprofen.

The spectrophotometric assessment of the active substance encapsulation degree was determined from the calibration curve. entrapping DEX <sup>4</sup> 0.636 <sup>1470</sup> 61.7 Very good stability

**(mV) Stability Criterion**

**Z Potential** 

For obtaining the release kinetics curve, we took into consideration that the medium volume remained constant over time (solvent evaporation during the experiment was negligible). The etalonation curve of variation of DEX concentration over time was built using the UV absorption spectra (Figure 4). The measurements were performed in the UV domain, at 259 nm, the maximum absorption spectrum for DEX, because neither the solvents, nor the other polymer ingredients used, absorbed at the given wavelength. liposomes entrapping DEX 6.89 0.435 <sup>699</sup> 3.89 Threshold of light dispersion DEX: dexketoprofen. *3.2. In Vitro Release of DEX from Vesicles Stabilized with Chitosan* 

> The analysis of the in vitro release curve revealed a slower release of DEX from chitosan-coated liposomes compared to the release from DEX solution. Detailing the aspects of the drug kinetic profile, it was found that, after one hour, 43% of DEX was released from DEX crystals, while only 20% was released from the nanovesicles. Drug release reached a percentage of 74% from DEX solution, and 41% from the nano-DEX after three hours. Finally, the DEX release from nanovesicles appeared to be effective, reaching a maximum of 92% after eight hours, while almost 80% of active substance was released from the DEX solution (Figure 5). The lower percentage of the drug released from the nano-DEX was attributed to the high dispersion of the active substance as isolated molecules well-entrapped into vesicles stabilized with chitosan. The spectrophotometric assessment of the active substance encapsulation degree was determined from the calibration curve. For obtaining the release kinetics curve, we took into consideration that the medium volume remained constant over time (solvent evaporation during the experiment was negligible). The etalonation curve of variation of DEX concentration over time was built using the UV absorption spectra (Figure 4). The measurements were performed in the UV domain, at 259 nm, the maximum absorption spectrum for DEX, because neither the solvents, nor the other polymer ingredients used, absorbed at the given

**Figure 4.** The absorption spectra of DEX and nano-DEX. DEX: dexketoprofen; nano-DEX: lipo-**Figure 4.** The absorption spectra of DEX and nano-DEX. DEX: dexketoprofen; nano-DEX: liposomes containing dexketoprofen.

The analysis of the in vitro release curve revealed a slower release of DEX from chitosan-coated liposomes compared to the release from DEX solution. Detailing the aspects of the drug kinetic profile, it was found that, after one hour, 43% of DEX was released from DEX crystals, while only 20% was released from the nanovesicles. Drug release reached a percentage of 74% from DEX solution, and 41% from the nano-DEX after three hours. Finally, the DEX release from nanovesicles appeared to be effective, reaching a maximum of 92% after eight hours, while almost 80% of active substance was released from the DEX solution (Figure 5). The lower percentage of the drug released from the nano-DEX was attributed to the high dispersion of the active substance as isolated

molecules well-entrapped into vesicles stabilized with chitosan.

**Figure 5.** The release kinetics of DEX from nano-DEX *vs*. DEX solution by the permeation method. DEX: dexketoprofen; nano-DEX: liposomes containing dexketoprofen. **Figure 5.** The release kinetics of DEX from nano-DEX *vs*. DEX solution by the permeation method. DEX: dexketoprofen; nano-DEX: liposomes containing dexketoprofen.

#### *3.3. The In Vivo Biocompatibility Testing 3.3. The In Vivo Biocompatibility Testing*

No significant differences in the percentage of the blood count formula elements between DEX, nano-DEX and the DW group were observed during the experiment (Table 2). No significant differences in the percentage of the blood count formula elements between DEX, nano-DEX and the DW group were observed during the experiment (Table 2).

**Table 2.** The effects of DEX and nano-DEX on the percentage of the blood count formula elements (values are expressed as mean ± S.D. for 6 rats in a group). **Table 2.** The effects of DEX and nano-DEX on the percentage of the blood count formula elements (values are expressed as mean ± S.D. for 6 rats in a group).


morphonuclear neutrophils, PMN; lymphocytes, Ly; eosinophils, E; monocyte, M; basophils, B. The treatment with DEX and nano-DEX did not induce major variations in the AST, DW: distilled water; DEX: dexketoprofen; nano-DEX: liposomes containing dexketoprofen; polymorphonuclear neutrophils, PMN; lymphocytes, Ly; eosinophils, E; monocyte, M; basophils, B.

ALT and LDH activity, compared to control, after one day, or seven days (Table 3). The treatment with DEX and nano-DEX did not induce major variations in the AST, ALT and LDH activity, compared to control, after one day, or seven days (Table 3).

**Table 3.** The effects of DEX and nano-DEX on the AST, ALT and LDH activity (values are expressed as mean ± S.D. for 6 rats in a group). **AST (U/mL) ALT (U/mL) LDH (U/L)** The use of DEX and nano-DEX did not show substantial modifications in the serum level of urea and creatinine compared to the control group, at both time points of the determinations (Table 4).

DW 1 day 54.5 ± 6.7 67.3 ± 8.3 336.42 ± 70.17 7 days 52.3 ± 5.9 68.1 ± 9.3 346.29 ± 71.45 DEX 1 day 54.2 ± 6.3 66.5 ± 7.5 341.33 ± 71.67 No considerable variations were revealed in the serum complement activity, nor in the PC between DEX, nano-DEX groups, and control, one day or seven days after substance administration (Table 5).

nano-DEX 1 day 53.7 ± 5.5 66.1 ± 8.3 356.72 ± 69.55

DW: distilled water; DEX: dexketoprofen; nano-DEX: liposomes containing dexketoprofen; aspar-

tate transaminase, AST;alanine aminotransferase, ALT; lactate dehydrogenase, LDH.

7 days 56.5 ± 7.1 69.3 ± 9.1 353.37 ± 69.29

7 days 55.3 ± 6.5 68.7 ± 8.7 360.29 ± 68.13


**Table 3.** The effects of DEX and nano-DEX on the AST, ALT and LDH activity (values are expressed as mean ± S.D. for 6 rats in a group).

DW: distilled water; DEX: dexketoprofen; nano-DEX: liposomes containing dexketoprofen; aspartate transaminase, AST;alanine aminotransferase, ALT; lactate dehydrogenase, LDH.

**Table 4.** The effects of DEX and nano-DEX on the blood level of urea and creatinine (values are expressed as mean ± S.D. for 6 rats in a group).


DW: distilled water; DEX: dexketoprofen; nano-DEX: liposomes containing dexketoprofen.

**Table 5.** The effects of DEX and nano-DEX on the complement activity and on the PC (values are presented as mean ± S.D. for 6 rats in a group).


DW: distilled water; DEX: dexketoprofen; nano-DEX: liposomes containing dexketoprofen; PC: phagocytic capacity of peripheral neutrophils.
