# The Anti-Arthritic Activity of Diclofenac Lipid-Core Nanocapsules: Stereological Analysis Showing More Protection of Deep Joint Components

^{1}

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## Abstract

**:**

## 1. Introduction

## 2. Results

#### 2.1. Preparation of DIC-LNC Dispersed in Water

^{12}and 4.98 × 10

^{12}nanocapsules mL

^{−1}(Table 1).

^{−1}and encapsulation efficiency of 100%. The pH and zeta potential were determined, and no significant differences were observed between DIC-LNCs and LNC.

#### 2.2. DIC-LNC Reduces Edema Formation at the Hind Paws

#### 2.3. DIC-LNC Reduces the Serum Levels of Proinflammatory Cytokines and CRP

#### 2.4. Absence of Liver and Renal Toxicity with DIC-LNC

#### 2.5. Cavalieri’s Volume of Metatarsophalangeal (MTP) Joints

#### 2.6. Density of the Joint Components and Absolute Volume

#### 2.7. Surface Area of MTP Joints

#### 2.8. Quantification of Chondrocytes

## 3. Discussion

## 4. Materials and Methods

#### 4.1. Materials

^{−1}) and sorbitan monostearate (Span 60

^{®}) were supplied by Sigma-Aldrich Co. (Burlington, VT, USA). Caprylic/capric triglyceride (CCT) was obtained from Delaware (Brazil), and polysorbate 80 from Brasquim (Brazil). Sodium diclofenac was purchased from Galena (Brazil). All other chemicals and solvents were of analytical or pharmaceutical grade. All the reagents were used as received.

#### 4.2. Preparation of the Lipid-Core Nanocapsules Formulations

^{−1}of hydrochloric acid until turbidity was observed. The mixture was kept static to allow precipitation in a cooling bath, and the precipitate obtained was filtered and recrystallized using water: ethanol (1:1, v/v). The colorless crystals were characterized by infrared spectroscopy (FT-IR 8300, Shimadzu) with a resolution of 4 cm

^{−1}and 64 scans, and the range of the frequency from 4000 to 500, presenting bands at wavelength numbers (cm

^{−1}) of 3300 (NH), 3200–2500 (OH), and 1710 (C=O).

#### 4.3. Physicochemical Characterization of the Formulations

#### 4.3.1. pH Measurements

#### 4.3.2. Electrophoretic Mobility and Zeta Potential

^{−1}NaCl aqueous solution (1:500, v/v), and the analysis was carried out at 25 °C.

#### 4.3.3. Laser Diffraction

#### 4.3.4. Dynamic Light Scattering

^{®}), and the intensity of the scattered light was analyzed at an angle of 173°, providing correlation curves, which were fitted using a monoexponential model.

#### 4.3.5. Nanoparticle Tracking Analysis

^{®}). Based on the Brownian motion of the lipid-core nanocapsules, the hydrodynamic diameter (Dh), median diameter (D50), diameter at the 90th percentile (D90) under the cumulative size distribution, and particle number density (PND) were obtained in real time using a CCD camera. The video clips were captured over 60 s at 21.6 ± 0.5 °C and 0.96 ± 0.02 cP.

#### 4.3.6. Drug Content and Encapsulation Efficiency

^{−1}and injection volume of 20 μL. The sample was diluted in acetonitrile in a volumetric flask and filtered with 0.45 μm membranes (Millipore, Burlington, MA, USA). The methodology presented proper linear regression, with r > 0.999 in the interval used, 1–50 μg mL

^{−1}, and demonstrated specificity, accuracy (99 ± 1%), repeatability, and precision (relative standard deviation <5%).

^{®}centrifugal filter device (10 KDa, Millipore

^{®}USA), after which the unit was centrifuged at 1844× g (RCF) for five min using a centrifuge (Sigma

^{®}1-14, Osterode, Germany). The ultrafiltrate was analyzed by HPLC as described above. The EE% was calculated as the difference between the total drug content (total concentration of the drug in the formulation) and the drug concentration in the ultrafiltrate (concentration of the dissolved drug in the continuous phase), divided by the total content, and multiplied by 100. All analyses were performed in triplicate.

#### 4.4. Animals

#### 4.5. Induction of Arthritis by Freund’s Complete Adjuvant and Experimental Design

^{TM}, Franklin Lakes, NJ, USA) into the tail of the rats after inhalation of isoflurane anesthesia on the first day of the experiment. After evidence of arthritis, the animals were allocated into four groups using simple randomization: Group 1, control (arthritic rats without treatment); Group 2, arthritis with empty LNC; Group 3, arthritis with a free solution of diclofenac; and Group 4, arthritis with the nanoformulation DIC-LNCs. On day 0 of the experiment, each treatment was initiated with an intraperitoneal injection of DIC, DIC-LNC, and empty LNC, respectively, for each group. The DIC and DIC-LNC doses were 3 mg/kg/day. On day 28 of the experiment, the animals were euthanized by isoflurane inhalation, and blood was obtained by heart puncture.

#### 4.6. Randomization, Blinding, and Allocation Concealment

#### 4.7. Edema Volume and Arthritis Score of the Hind Paw

^{®}, Ribeirão Preto, Brazil). A blinded evaluator assessed paw volume. The arthritis score was evaluated on the 28th day of the experiment. Rat paws were scored using an arthritis score as previously described [48].

#### 4.8. Quantification of Cytokines and Biochemical Markers

^{®}(cytometry bead array), the serum levels of TNF-α, IL-1α, and CRP were measured, following the manufacturer’s instructions. A FACSCalibur flow cytometer (BD Biosciences) was used to analyze the samples. The quantity and mean fluorescence of the cytokines were calculated using BD FCAP Array™ Software (v 1.0.1; BD Biosciences). A Roche Hitachi Chemistry Analyzer and an immunoturbidimetric assay (catalog number: 4956842190) were used to calculate the serum levels of CRP, following the manufacturer’s instructions. Liver and renal toxicities were evaluated by measuring glutamic oxaloacetic transaminase (TGO), glutamic pyruvic transaminase (TGP), gamma-glutamyl transpeptidase (GGT, creatinine, and blood urea nitrogen (BUN).

#### 4.9. Stereological Analysis

^{+}borate, 0.5 g; distilled H

_{2}O, 100 mL) and basic fuchsin (basic fuchsin, 0.5 g and distilled H

_{2}O, 100 mL).

#### 4.10. Determination of Cavalieri’s Volume

^{2}), and T (50 µm) was the distance between each section [50].

#### 4.11. Determination of Relative Volume

#### 4.12. Determination of Surface Area

#### 4.13. Counting the Number of Cellular Profiles

^{2}), each containing a reference point. The counting frames have a solid forbidden line and a dashed acceptance line. Only the nuclei of cells that appeared inside the counting frame and did not touch the exclusive line were counted. The number of cells was expressed as profiles/mm

^{2}and calculated using the following formula:

#### 4.14. Statistical Analysis

## 5. Conclusions

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Conflicts of Interest

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**Figure 1.**Arthritis evaluation and scores for twenty-eight days. (

**A**) Arthritis kinetics, the black arrows indicate the maximum effects observed. (

**B**) Edema curve with smoothing techniques. (

**C**) Arthritis score. For convenience, at the figures the number of asterisks over the bars are the number of digits with significance after the decimal point (* significance at one digit after decimal point).

**Figure 2.**Serum inflammatory markers on day twenty-eight of the experiment. (

**A**) Quantification of the proinflammatory cytokines TNF-α and IL-1α. (

**B**) Serum levels of CRP for the DIC and DIC-LNC groups. (

**C**) xanthine-oxidase as an oxidative stress marker. For convenience, at the figures the number of asterisks over the bars are the number of digits with significance after the decimal point (* significance at one digit after decimal point; ** significance at two digits after decimal point; *** significance at three digits after decimal point; **** significance at four digits after decimal point).

**Figure 3.**Liver and kidney toxicity biochemical markers. (

**A**) Liver biochemical markers (TGO, TGP, GGT) of all five groups (Ul/mL). (

**B**) Kidney biochemical markers (BUN, CRE) for all five groups (mg/dL).

**Figure 4.**Relationship between Cavalieri’s volume and the coefficient of error. Demonstration of the relation between the number of serial sections (x-axis), the volume of Cavalieri (right y-axis), and the coefficient of error (left y-axis). Continuous lines indicate the volume, and dotted lines represent the error coefficient. Blue indicates 28 equidistant 20 mm serial sections; purple indicates 14 equidistant 40 mm serial sections; pink indicates 10 equidistant 60 mm serial sections; orange line indicates 16 equidistant 80 mm serial sections.

**Figure 5.**The volume of the MTP joint and each component. (

**A**) MTP joint volume of all treated groups. (

**B**) Absolute volume of the components of MTP of each treated group. (

**C**) Volume of Capsule, Synovial Space, and Synovial Membrane. ANOVA significance p < 0.05.

**Figure 6.**The surface area of MTP. The left panel presents the surface area of the cartilage. The right panel presents the surface area of the synovial membrane. ANOVA significance p < 0.05. For convenience, at the figures the number of asterisks over the bars are the number of digits with significance after the decimal point (* significance at one digit after decimal point; ** significance at two digits after decimal point; *** significance at three digits after decimal point; **** significance at four digits after decimal point).

**Figure 7.**Cellular profile count of MTP chondrocytes. The left panel shows the total number of chondrocytes count. The middle panel shows the number of isolated chondrocytes. The right panel shows the number of isogenous groups of chondrocytes present after mitosis. ANOVA significance p < 0.05. For convenience, at the figures the number of asterisks over the bars are the number of digits with significance after the decimal point (* significance at one digit after decimal point; *** significance at three digits after decimal point; **** significance at four digits after decimal point).

**Figure 8.**Methods used for stereological analysis. (1) Sectioning level of the 2nd digit of the paw. (2) Block of resin containing an MTP joint (longitudinal lines indicate the location of the knife during the microtomy. Generation of 50 μm equidistant serial sections. Any section passing through the longitudinal plane of the finger will be a vertical section. (3) Profile of a section used to determine Cavalieri volume. In this magnification, the structures marked in the image are fully visible. The lines delimit the area investigated in this study limited by the border with the spongy bone. Detail of the joint region used to estimate the volume density (point-counting system), surface area (counting system with points and curves), and several chondrocyte profiles (counting system with frames and a central point). cap, capsule; b, bone; ca, cartilage; s, synovial space. (4) Integrative equations for quantitative stereology.

**Table 1.**Characterization of the formulations by potentiometry, laser diffraction, dynamic light scattering, and nanoparticle tracking analysis.

LNC | DIC-LNC | |
---|---|---|

Potentiometry | ||

pH | 5.43 ± 0.24 | 5.39 ± 0.16 |

Laser diffraction | ||

D[4,3] (nm) | 153 ± 10 | 204 ± 46 |

Span | 1.4 ± 0.2 | 1.7 ± 0.1 |

Dynamic Light Scattering | ||

Dh (nm) | 170 ± 13 | 166 ± 13 |

PDI | 0.06 ± 0.02 | 0.08 ± 0.02 |

Electrophoretic mobility | ||

Zeta Potential (mV) | −13 ± 6 | −11 ± 2 |

Nanoparticle tracking analysis | ||

Dh (nm) | 182 ± 9 | 196 ± 14 |

D50 (nm) | 173 ± 13 | 186 ± 2 |

D90 (nm) | 257 ± 10 | 309 ± 9 |

PND (× 10^{12} particles mL^{−1}) | 4.98 ± 0.25 | 4.76 ± 0.78 |

Volume (mm^{3}) | ||||||
---|---|---|---|---|---|---|

Groups | Joint | Cartilage | Bone | Capsule | Synovial Space | Synovial Membrane |

Arthritis | 3.02 ± 0.08 | 0.50 ± 0.10 | 1.20 ± 0.05 | 0.97 ± 0.14 | 0.16 ± 0.01 | 0.08 ± 0.02 |

LNC | 2.80 ± 0.32 | 0.36 ± 0.08 | 1.08 ± 0.16 | 0.89 ± 0.15 | 0.17 ± 0.01 | 0.04 ± 0.01 |

Diclofenac | 2.46 ± 0.12 | 0.34 ± 0.03 | 1.02 ± 0.12 | 0.67 ± 0.02 | 0.19 ± 0.02 | 0.03 ± 0.00 |

Diclofenac-LNC | 2.10 ± 0.10 | 0.21 ± 0.01 | 0.75 ± 0.05 | 0.58 ± 0.02 | 0.26 ± 0.01 | 0.02 ± 0.00 |

No Arthritis | 1.95 ± 0.05 | 0.23 ± 0.03 | 0.61 ± 0.06 | 0.52 ± 0.03 | 0.41 ± 0.03 | 0.01 ± 0.05 |

Surface Area (mm^{2}) | Cellular Count (Cell/mm^{2}) | ||||
---|---|---|---|---|---|

Groups | Cartilage | Synovial Membrane | Chondrocytes | Isogenous Groups | Total Chondrocytes |

Arthritis | 38.25 ± 4.32 | 13.26 ± 2.78 | 1416 ± 126.3 | 80.55 ± 8.77 | 1517 ± 54.30 |

LNC | 29.09 ± 3.75 | 9.98 ± 1.49 | 1543 ± 49.92 | 111.5 ± 4.51 | 1701 ± 91.96 |

Diclofenac | 27.87 ± 3.31 | 9.92 ± 0.46 | 1627 ± 95.61 | 161.1 ± 11.84 | 1849 ± 16.73 |

Diclofenac-LNC | 20.45 ± 1.58 | 8.01 ± 0.19 | 1894 ± 47.00 | 226.8 ± 20.5 | 2095 ± 52.33 |

No Arthritis | 19.45 ± 0.28 | 7.43 ± 1.04 | 2208 ± 217.4 | 297.9 ± 19.5 | 2367 ± 225.8 |

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**MDPI and ACS Style**

Ureña, N.M.; de Oliveira, C.P.; Guterres, S.S.; Pohlmann, A.R.; da Costa, O.T.F.; Boechat, A.L.
The Anti-Arthritic Activity of Diclofenac Lipid-Core Nanocapsules: Stereological Analysis Showing More Protection of Deep Joint Components. *Molecules* **2023**, *28*, 5219.
https://doi.org/10.3390/molecules28135219

**AMA Style**

Ureña NM, de Oliveira CP, Guterres SS, Pohlmann AR, da Costa OTF, Boechat AL.
The Anti-Arthritic Activity of Diclofenac Lipid-Core Nanocapsules: Stereological Analysis Showing More Protection of Deep Joint Components. *Molecules*. 2023; 28(13):5219.
https://doi.org/10.3390/molecules28135219

**Chicago/Turabian Style**

Ureña, Nathalie Marte, Catiúscia Padilha de Oliveira, Silvia Stanisçuaski Guterres, Adriana Raffin Pohlmann, Oscar Tadeu Ferreira da Costa, and Antonio Luiz Boechat.
2023. "The Anti-Arthritic Activity of Diclofenac Lipid-Core Nanocapsules: Stereological Analysis Showing More Protection of Deep Joint Components" *Molecules* 28, no. 13: 5219.
https://doi.org/10.3390/molecules28135219