# The Physicochemical, Biopharmaceutical, and In Vitro Efficacy Properties of Freeze-Dried Dexamethasone-Loaded Lipomers

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## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Materials and Lipomer Formulation

#### 2.2. Freeze Drying

#### 2.3. Physicochemical Characterization

#### 2.3.1. Z-Average, PdI, and Z-Potential before and after Freeze Drying

#### 2.3.2. Transmission Electron Microscopy before and after Freeze Drying

#### 2.3.3. Differential Scanning Calorimetry before and after Freeze Drying

#### 2.4. Gel Formulations

^{−1}.

_{i}denotes the empirical shear rate, and y

_{i}denotes the empirical shear stress.

#### 2.5. High-Performance Liquid Chromatography (HPLC)

_{2}PO

_{4}0.05M (60:40) passed at 1.8 mL/min through a C18 HPLC column (250 × 4.6 mm, 3 µm) at 25 °C. The detection wavelength was 208 nm, and the injection volume was 20 µL. The assay was performed with an HPLC instrument (Waters 2695 and detector Waters 2996, Waters Corporation, Milford, MA, USA). In addition, this method was used to estimate the encapsulation efficiency (%EE) according to Equation (8), where WT denotes the total content of DEX in the formulation and W

_{NE}denotes the DEX obtained in the filtrate (not encapsulated) after centrifugation of the amicon ultra device (Merck Millipore, Barcelona, Spain) with a membrane cut-off of 100 KDa at 4500 rpm for 30 min.

#### 2.6. In Vitro Release Tests

^{2}. Experimental variables were the same as previously used [7]: briefly, receptor medium (ethanol:purified water 50:50) at 32 °C, 60 mg of DEX placed in the donor compartment, and 0.3 mL of sample volume obtained at regular time intervals of up to 24 h. A 12–14 KDa dialysis membrane (Spectrum Chemical, New Brunswick, NJ, USA) was placed between the donor and receptor compartments. Model fitting to several kinetic equations (Table 2) was performed with the DD-solver Excel add-in [19] using the lowest Akaike Information Criteria (AIC) as the model selection criteria.

#### 2.7. Pig Skin In Vitro Permeation Tests

_{sup}denotes the transdermal flux in a steady state, Q

_{t}denotes the permeated amount at time t, t denotes the time, s denotes the diffusional area, K

_{p}denotes the permeability coefficient, C

_{d}denotes the concentration of the drug in the donor compartment, P

_{1}denotes the diffusion parameter, P

_{2}denotes the partitioning parameter, and t

_{lag}denotes the lag time.

_{lag}was estimated as the extrapolation in the x-axis (x-intercept) of the plot cumulative amounts vs. time.

#### 2.8. In Vitro Cytotoxicity/Anti-TNFα Efficacy

_{2}. The HEK001 cell medium was a Keratinocyte Serum Free supplemented with Epidermal Growth Factor (EGF; 100 µg/mL) (Life Technologies, Carlsbad, CA, USA), penicillin (20 U/mL), and streptomycin (Life Technologies; 20 μg/mL). HaCaT was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Gln) (Life Technologies), 20 U/mL penicillin, and 20 μg/mL streptomycin (Life Technologies). A PCR amplification was carried out every 14 days to confirm the absence of Mycoplasma contamination. Both cell lines, HEK001 and HaCaT, were used to test the cytotoxicity and the anti-inflammatory effect of the formulations.

#### 2.9. Statistical Analysis

## 3. Results and Discussion

#### 3.1. Freeze Drying

#### 3.2. Physicochemical Characterization before and after Freeze Drying

#### 3.3. Gel Formulations Rheology Studies

^{−1}(area 0.92 ± 0.38). In addition, when shear stress ended, the internal structure did not completely recover, probably because more time was required for this to occur. To ascertain the rheology behavior, experimental data were fit to different equations. The equation costs are shown in Table 5. The lower cost represents the best fit.

_{0}) was required to start the flow, below which the value the formulation acted like a solid. The flow behavior changed when lipomers were loaded into the hydrogel. Yield stress was almost null. A similar cost function value was also observed for the Herschel–Bulkley and Ostwald–de Waele models (which is the same model but without yield stress). The n value was lower than 1 in all cases, which represents a pseudoplastic profile. This profile was less predominant in the DEX-lipomer hydrogel, followed by the DEX hydrogel.

#### 3.4. In Vitro Release Tests

#### 3.5. In Vitro Permeation Tests

_{p}or J

_{sup}. Although there were differences in the maximum permeated drug concentration, the slope of both formulations was similar, and the same transdermal flux was obtained. The significance of P

_{1}(diffusion-related parameter) was borderline (p = 0.08), which was probably related with the differences in drug release; however, the power of the statistical test was not enough to discriminate between both formulations. Clear statistical differences were found in lag time and related parameters (diffusion related parameter, P

_{2}). The FD process increased the lag time, which was probably caused by the increase in particle size (double that of non-FD particles) and the modification of nanoparticle structure. The greater particle size can affect the number of nanoparticles that are stored in the hair follicles and may reduce the packaging of the particles in the skin surface, i.e., the greater the size, the lower the surface-to-volume ratio. The modification of the lag time brings about the modification of P

_{2}(the partitioning-related parameter), which is reduced compared with the non-FD formulation. This could be caused by the reduced amount of intimate contact between both systems (due to the increase in particle size and the reduction in the surface-to-volume ratio), which may lead to a decrease in P

_{2}. Another possibility is that the cryoprotectant modifies the formulation/skin partitioning, because these compounds can form hydrogen bonds with the nanoparticle surface and alter the interaction with the skin.

#### 3.6. Cytotoxicity and Anti-TNFα Efficacy

## 4. Conclusions

## Supplementary Materials

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

## References

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**Figure 2.**FDM pictures of a drop of DEX-lipomers (no cryoprotectant) showing different thermal events: (

**A**) liquid sample; (

**B**) freezing; (

**C**) sublimation front; (

**D**) collapse temperature.

**Figure 3.**TEM picture of lipomers after negative staining with uranyl acetate. Panel (

**A**) corresponds to non-FD lipomers (image reproduced from [7], MDPI, 2021). Panel (

**B**) corresponds to FD lipomers.

**Figure 4.**DSC thermogram of non-FD DEX-lipomers (blue curve), FD DEX-lipomers (green curve), and DEX (red curve).

**Figure 7.**DEX release percentages of the tested formulations (n = 6). Results shown mean and standard deviation values.

**Figure 8.**Transdermal DEX (lipomers before and after lyophilization) profile in dermatomed pig skin (n = 6). The results show the mean and standard deviation.

**Figure 9.**Cytotoxicity and anti-TNFα efficacy studies in HEK001 (

**A**,

**C**) and HaCaT cells (

**B**,

**D**). (

**A**,

**B**) Cell viability analyzed by MTT. The indicated numbers represent the inverse dilution factors, referring to the synthetized lipomers. Dilution factors for DEX-loaded lipomers correspond to concentrations of 5, 1, 0.5, 0.25, and 0.1 μM of DEX. Data are represented as the mean ± SEM (n = 3) of the cell viability percentage, referring to untreated controls (horizontal lane). Statistical significance was assessed by two-way ANOVA ** p < 0.01; **** p < 0.001. (

**C**,

**D**) TNFα mRNA expression was determined after a 24 h treatment of free DEX 0.1 μM (black), non-FD DEX-lipomers (gray), or FD DEX-lipomers (dark gray), without (

**left**) or with (

**right**) a 1 h pretreatment with LPS (10 μg/mL). TNFα expression is represented as the mean ± SEM (n = 4 HEK001; n = 3 HaCaT); horizontal pink and red lanes represent TNFα expression under control and LPS conditions, respectively. Statistical significance was evaluated by one-way ANOVA compared to LPS; * p < 0.05, ** p < 0.01.

Rheological Model | Equation | |
---|---|---|

Newton | $\tau =\eta \xb7\dot{\gamma}$ | (1) |

Bingham | $\tau ={\tau}_{0}+({\eta}_{0}\xb7\dot{\gamma )}$ | (2) |

Ostwald–de Waele | $\tau =K\xb7{\dot{\gamma}}^{n}$ | (3) |

Herschel–Bulkley | $\tau ={\tau}_{0}+K\xb7{\dot{\gamma}}^{n}$ | (4) |

Casson | $\tau =\sqrt[n]{\left({\tau}_{0}^{n}+{\left({\eta}_{0}\xb7\dot{\gamma}\right)}^{n}\right)}$ | (5) |

Cross | $\tau =\dot{\gamma}\xb7({\eta}_{\infty}+({\eta}_{0}-{\eta}_{\infty})/(1+{(\dot{\gamma}/{\dot{\gamma}}_{0})}^{n})$ | (6) |

_{0}denotes the yield stress (Pa), η

_{0}denotes the zero-shear viscosity (Pa∙s), η

_{∞}denotes the infinite-shear viscosity, K denotes the consistency index, n denotes the flow index, and ${\dot{\gamma}}_{0}$ denotes the zero-shear rate (1/s).

Kinetic Model | Equation | |
---|---|---|

First Order | $F={F}_{max}\text{}\left(1-{e}^{\left(-{K}_{1}t\right)}\right)$ | (9) |

Higuchi | $F={K}_{H}\text{}\xb7\text{}{t}^{\frac{1}{2}}$ | (10) |

Korsmeyer–Peppas | $F={K}_{\mathrm{KP}}\text{}\xb7\text{}{t}^{n}$ | (11) |

Weibull | $F=1-{e}^{\left(\raisebox{1ex}{$-t$}\!\left/ \!\raisebox{-1ex}{${T}_{d}$}\right.\right)\beta}$ | (12) |

_{1}, K

_{H}, and K

_{KP}denote the release constant of the first-order, Higuchi, and Korsmeyer–Peppas (KP) functions, respectively; T

_{d}denotes the time required to dissolve the 63.2% of the drug dose; and β denotes the shape parameter of the Weibull function. The value of exponent n of KP describes the release mechanism (n < 0.43 represents a Fickian diffusion; 0.43 ≤ n ≤ 0.85 corresponds to anomalous transport; n > 0.85 corresponds to a case II transport).

FD Cycle | Temperature | Time |
---|---|---|

Soak | 10 °C | 1 h |

Freezing | −55 °C | 4 h |

Primary Drying | −30 °C | 72 h |

Secondary Drying ramp | −30 °C to 30 °C | 4 h |

Secondary Drying | 30 °C | 4 h |

**Table 4.**Particle size, polydispersion (PDI), zeta potential, and encapsulation efficiency (EE) of nanoparticles before and after lyophilization with different cryoprotectants.

Formulation | Before Freeze Drying | After Freeze Drying | ||||||
---|---|---|---|---|---|---|---|---|

Hydrodynamic Diameter (nm) | PDI | Z-Pot (mV) | EE (%) | Hydrodynamic Diameter (nm) | PDI | Z-Pot (mV) | EE (%) | |

DEX-lipomers (no cryo) | 185.23 ± 5.24 | 0.360 ± 0.019 | 39.0 ± 0.1 | 98.60 ± 0.01 | 1850.00 ± 188.75 | 0.313 ± 0.051 | 35.9 ± 2.0 | 98.94 ± 0.01 |

DEX-lipomers (trehalose 6%) | 186.87 ± 2.68 | 0.361 ± 0.015 | 36.3 ± 0.4 | 446.70 ± 3.21 | 0.355 ± 0.013 | 34.3 ± 0.5 | 98.97 ± 0.01 | |

DEX-lipomers (sucrose 6%) | 185.67 ± 4.92 | 0.349 ± 0.016 | 37.3 ± 0.5 | 374.33 ± 7.60 | 0.229 ± 0.011 | 34.7 ± 0.4 | 98.87 ± 0.01 | |

DEX-lipomers (mannitol 6%) | 183.97 ± 1.27 | 0.334 ± 0.008 | 37.4 ± 0.4 | 749.53 ± 26.49 | 0.435 ± 0.013 | 34.9 ± 1.7 | 94.63 ± 6.22 |

**Table 5.**Rheological model fitting of hydrogel placebo, dexamethasone hydrogel, and freeze-dried dexamethasone lipomers hydrogel.

Rheological Model | Hydrogel Placebo (Cost) | FD-DEX-Lipomers Hydrogel (Cost) | DEX Hydrogel (Cost) |
---|---|---|---|

Newton | 904.563 | 137.743 | 768.095 |

Bingham | 59.985 | 19.360 | 44.060 |

Ostwald–de Waele | 23.138 | 0.665 | 13.814 |

Herschel–Bulkley | 2.721 | 0.656 | 3.372 |

Casson | 4.952 | 0.791 | 4.883 |

Cross | 2.080 | 0.142 | 2.529 |

**Table 6.**Rheological parameters of the Herschel–Bulkley equation of hydrogel placebo, DEX hydrogel, and FD DEX-lipomers hydrogel (mean ± standard deviation). (*) denoted statistical differences p < 0.05.

Herschel–Bulkley Equation Parameter | Hydrogel Placebo | DEX-Lipomers Hydrogel | DEX Hydrogel |
---|---|---|---|

${\tau}_{0}$ (Pa) | 11.312 ± 1.990 | −0.120 ± 0.250 * | 11.283 ± 0.189 |

K | −2.040 ± 0.957 | 2.971 ± 0.523 * | −2.935 ± 0.196 |

n | −0.611 ± 0.076 * | 0.259 ± 0.019 * | −0.411 ± 0.025 * |

**Table 7.**Viscoelasticity parameters of hydrogel placebo, dexamethasone hydrogel, and freeze-dried dexamethasone lipomers hydrogel (mean ± standard deviation (SD)). (*) denoted statistical differences p < 0.05.

Parameter | Formulation | Mean ± SD |
---|---|---|

G′ | Placebo gel | 17.48 ± 0.44 Pa |

Gel DEX-lipomer | 9.97 ± 0.35 Pa (*) | |

Gel DEX | 16.19 ± 0.13 Pa (*) | |

G″ | Placebo gel | 4.88 ± 0.09 Pa |

Gel DEX-lipomer | 5.55 ± 0.24 Pa (*) | |

Gel DEX | 4.60 ± 0.03 Pa | |

G* | Placebo gel | 18.16 ± 0.43 Pa |

Gel DEX-lipomer | 11.35 ± 0.42 Pa (*) | |

Gel DEX | 16.84 ± 0.12 Pa | |

tan δ | Placebo gel | 15.6 ± 0.64° |

Gel DEX-lipomer | 29.4 ± 0.09° (*) | |

Gel DEX | 15.8 ± 0.25° |

**Table 8.**Results of model fitting of DEX drug release for the prepared formulations. AIC in bold corresponds to the model that best fit the experimental data. “n” corresponds to the release exponent of Korsmeyer–Peppas (KP) equation.

Formulation | Model | AIC | Parameters | Value |
---|---|---|---|---|

Freeze-dried DEX-lipomers | First order | 28.59 | k (h^{−1}) | 0.046 |

Higuchi | 57.87 | k_{H} (%h^{−1/2}) | 12.579 | |

Korsmeyer–Peppas | 35.48 | n | 0.800 | |

k_{KP} (%h^{−n}) | 5.261 | |||

Weibull | 28.57 | t_{d} (h) | 22.75 | |

β | 1.008 | |||

Freeze-dried DEX-lipomers hydrogel | First order | 44.988 | k (h^{−1}) | 0.155 |

Higuchi | 46.539 | k_{H} (%h^{−1/2}) | 44.196 | |

Korsmeyer–Peppas | 49.847 | n | 0.383 | |

k_{KP} (%h^{−n}) | 10.960 | |||

Weibull | 48.870 | t_{d} (h) | 11.960 | |

β | 0.601 | |||

DEX hydrogel | First order | 48.877 | k (h^{−1}) | 0.133 |

Higuchi | 44.710 | k_{H} (%h^{−1/2}) | 11.277 | |

Korsmeyer–Peppas | 52.096 | n | 0.590 | |

k_{KP} (%h^{−n}) | 9.060 | |||

Weibull | 48.484 | t_{d} (h) | 10.965 | |

β | 0.700 |

Parameter | Non-Freeze-Drying DEX-Lipomers (Mean ± SD) | Freeze-Drying DEX-Lipomers (Mean ± SD) |
---|---|---|

J_{sup} (µg/h·cm^{2}) | 0.4759 ± 0.1123 | 0.3789 ± 0.2093 |

K_{p} (cm/h) | 7.9316 × 10^{−5} ± 1.900 × 10^{−5} | 6.3148 × 10^{−5} ± 5.156 × 10^{−5} |

P_{2} (1/h) | 0.0385 ± 0.0011 | 0.0117 ± 0.0039 (*) |

P_{1} (cm) | 0.0020 ± 0.0006 | 0.0073 ± 0.0067 |

t_{lag} (h) | 4.213 ± 0.064 | 11.978 ± 4.776 (*) |

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**MDPI and ACS Style**

Pena-Rodríguez, E.; Mata-Ventosa, A.; Garcia-Vega, L.; Pérez-Torras, S.; Fernández-Campos, F.
The Physicochemical, Biopharmaceutical, and In Vitro Efficacy Properties of Freeze-Dried Dexamethasone-Loaded Lipomers. *Pharmaceutics* **2021**, *13*, 1322.
https://doi.org/10.3390/pharmaceutics13081322

**AMA Style**

Pena-Rodríguez E, Mata-Ventosa A, Garcia-Vega L, Pérez-Torras S, Fernández-Campos F.
The Physicochemical, Biopharmaceutical, and In Vitro Efficacy Properties of Freeze-Dried Dexamethasone-Loaded Lipomers. *Pharmaceutics*. 2021; 13(8):1322.
https://doi.org/10.3390/pharmaceutics13081322

**Chicago/Turabian Style**

Pena-Rodríguez, Eloy, Aida Mata-Ventosa, Laura Garcia-Vega, Sandra Pérez-Torras, and Francisco Fernández-Campos.
2021. "The Physicochemical, Biopharmaceutical, and In Vitro Efficacy Properties of Freeze-Dried Dexamethasone-Loaded Lipomers" *Pharmaceutics* 13, no. 8: 1322.
https://doi.org/10.3390/pharmaceutics13081322