# DOPE/CHEMS-Based EGFR-Targeted Immunoliposomes for Docetaxel Delivery: Formulation Development, Physicochemical Characterization and Biological Evaluation on Prostate Cancer Cells

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## Abstract

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_{50}at a concentration of 65.74 nM in the PC3 cell line and 28.28 nM in the DU145 cell line. Immunoliposome, in turn, for PC3 cells reached an IC

_{50}of 152.1 nM, and for the DU145 cell line, 12.60 nM, a considerable enhancement of cytotoxicity for the EGFR-positive cell line. Finally, the immunoliposome internalization was faster and greater than that of liposome in the DU145 cell line, with a higher EGFR overexpression. Thus, based on these results, it was possible to obtain a formulation with adequate characteristics of nanometric size, a high encapsulation of DTX and liposomes and particularly immunoliposomes containing DTX, which caused, as expected, a reduction in the viability of prostate cells, with high cellular internalization in EGFR overexpressing cells.

## 1. Introduction

^{®}(commercial drug), nanotechnology is a viable alternative to promote cancer selective delivery, reduce side effects and improve treatment efficacy. Thus, among the various types of nanosystems, liposomes, nanospheres with concentric lipid bilayers formed of phospholipids—which can be natural or synthetic—and biocompatible, biodegradable and non-immunogenic materials, stand out for the ability to load hydrophilic and lipophilic drugs. In order to increase the circulation half-life, pegylation is commonly used, since PEG forms a steric impediment in the external part of the vesicle and prevents recognition by reticulo-endothelial system [5,6,7].

## 2. Materials and Methods

#### 2.1. Preparation of pH-Sensitive Liposomes

#### 2.1.1. Box–Behnken Design

_{0}(where k is the number of factors and C

_{0}is the number of central points) [21]. A total of 15 experiments were performed at three levels: low, medium and high (−1, 0, +1), with three variables to choose the most appropriate composition formulation and test the correlation between dependent and independent variables, employing the minimum number of experiments. The central point was replicated three times. Formulation parameters and parameter levels were chosen based on preliminary studies [9,22]. The independent variables established were DOPE:CHEMS molar ratio (X

_{1}), lipid:DTX molar ratio (X

_{2}) and sonication time (X

_{3}). The dependent variables studied were encapsulation percentage (Y

_{1}), particle size (Y

_{2}) and polydispersity index (Y

_{3}). The ranges of formulation parameters and responses are shown in Table 1.

_{1}, X

_{2}and X

_{3}) was represented using a quadratic model of second-order polynomial regression, generated by Minitab

^{®}statistical software, expressed by the following Equation (1):

_{i}represents a dependent variable, b

_{0}is the intercept coefficient of the points, b

_{1}to b

_{33}are regression coefficients calculated from the observed experimental values, and X

_{1}, X

_{2}and X

_{3}are the independent variables selected before initial experiments. The terms X

_{1}X

_{2}, X

_{1}X

_{3}and X

_{2}X

_{3}represent the interaction effects, and the term X

_{i}

^{2}(i = 1, 2 or 3) indicates the quadratic effects [8,23,24]. The result was statistically validated by analysis of variance, by statistical significance of coefficients and R

^{2}values. Statistical analysis results were considered significant for p-values < 0.10.

#### 2.1.2. Functionalization of pH Sensitive Liposome with Cetuximab (Immunoliposome)

#### 2.2. Physicochemical Characterization of Liposomes and Immunoliposomes

#### 2.2.1. Particle Size, Polydispersity and Zeta Potential

#### 2.2.2. Encapsulation Efficiency

#### 2.2.3. Atomic Force Microscopy

#### 2.2.4. Fourier Transform Infrared Spectroscopy (FTIR)

^{−1}.

#### 2.2.5. Thermal Analysis

^{−1}, and around 10 mg of sample was used in the analysis. The sample was placed in sealed aluminum crucibles with pierced lids, and during the experiment the flow of nitrogen (70 mL/min) was constant [26].

#### 2.2.6. Powder X-ray Diffractometry

#### 2.2.7. In Vitro Release Study

#### 2.3. Antibody Functionalization Characterization

#### 2.3.1. Electrophoresis

#### 2.3.2. Thermophoresis

**°**C to 95

**°**C. Data were analyzed using Nano-Temper Analysis software 1.3.2.880.

#### 2.3.3. Indirect ELISA

_{2}SO

_{4}stop solution was added. The plate was read in a microplate reader with absorbance at 450 nm.

#### 2.4. Cell Studies

_{2}.

#### 2.4.1. Cellular Viability Assay

_{50}), the optical density of the negative control (untreated cells) was marked as 100% and the result was calculated from the concentration-effect curves [30].

#### 2.4.2. Cellular Internalization

^{5}cells/well) and incubated for 24 h at 37 °C under the same conditions, which was recommended for each cell line, PC3 and DU145. Then, the wells were washed and the cells incubated with the samples diluted in incomplete medium for 24 h at 37 °C. After treatment, the wells were washed with saline and the cells fixed with 1% paraformaldehyde. After 15 min, the wells were washed again with saline and 4′,6-diamidino-2-phenylindole (DAPI) solution was added, which was left to rest for 10 min. Then, the wells were again washed with saline and the coverslips were poured onto histology slides containing Fluoromount. The slides were kept at rest under refrigeration, protected from light, prior to viewing under a confocal microscope (Zeiss 5 Live, Zeiss, Germany, λexc = 488 nm, λem = 552 nm, with a 20 X objective) [11,25]. For flow cytometry, 5 × 10

^{5}cells/well were plated in 6-well microplates and incubated for 24 h at 37 °C under the same conditions mentioned. Then, the cells were washed and incubated with the samples diluted in incomplete medium for 4 and 24 h at 37 °C. The cells were trypsinized, centrifuged and resuspended in saline solution and then they were added with propidium iodide (Thermo Scientific, Pittsburg, PA, USA) (5 μL of 50 μg/mL solution) and submitted to analysis in flow cytometer (FACSCalibur, BD, New Jersey, USA), using λexc = 488 nm, λem = 530/30 nm, for Dio and λexc = 488 nm and λem = 670 nm for propidium iodide [11,25].

#### 2.5. Statistical Analysis

^{®}and Design-Expert

^{®}. The result was statistically validated by analysis of variance, by statistical significance of coefficients and R

^{2}values. Statistical analyses were considered significant for p-values < 0.01. Atomic force microscopy, cell internalization and indirect ELISA results were analyzed using GraphPad Prism 8.0.1. Atomic force microscopy was expressed as mean ± Standard Error of Mean (SEM). A p < 0.05 was considered statistically significant. The result of cell internalization by cytometry was analyzed by a two-way ANOVA test with a Bonferroni posttest. A p < 0.01 was considered statistically significant. The indirect ELISA was analyzed by a one-way ANOVA test with a Tukey posttest between samples. A p < 0.05 was considered statistically significant.

## 3. Results and Discussion

_{1}), docetaxel/lipid ratio (X

_{2}) and sonication time (X

_{3})—related to encapsulation efficiency (Y

_{1}) particle size (Y

_{2}) and index of polydispersity (PDI) (Y

_{3}). A response surface methodology was used to verify the effects of variations in these parameters, and to determine the best preparation condition. It is possible to observe great variation in the three analyzed responses: encapsulation efficiency (13.45–89.36%), particle size (114.1–292.9 nm) and PDI (0.200–0.480). These variations may be associated with the complex effects of each of the independent factors and interactions with each other. For better observation and understanding of these effects, surface response surface graphs were generated (Figure 1).

_{1}(DOPE/CHEMS ratio) on the encapsulation efficiency, and an opposite relationship of the variables X

_{2}(docetaxel/lipids ratio) and X

_{3}(sonication time) in relation to X

_{1}. The steep slope obtained by the sonication time denotes a negative effect and that the encapsulation efficiency is more sensitive to this factor. Heterogeneous variation of encapsulation efficiency, observed in Figure 1A–C and Table 2, is related to several factors causing different effects when observing the dependent factors. A higher ratio of docetaxel to lipids seems to promote greater vesicle organization, leading to smaller particle sizes, but with slightly lower encapsulation efficiency. The association between vesicle stabilization effects promoted by CHEMS and the drug interactions (of lipophilic nature) with the hydrocarbon chain seems to explain these influences. In a study using DOPE:CHEMS:DSPE-PEG 2000 for paclitaxel delivery, an encapsulation percentage of 90% was obtained, similar to two of our results (89.36% and 88.37%) [45].

_{1}(DOPE:CHEMS ratio) and X

_{2}(docetaxel/lipids ratio) causes a significant effect on particle size. Considering all interactions estimated in the conditions addressed, 80% of formulations presented in the present study are within the size range described in previous works as ideal (between 70 and 200 nm). The perturbation plot in Figure 2II shows a negative effect with a steep slope for the variable X

_{1}(DOPE:CHEMS ratio) on particle size. The sonication time (X

_{3}) also has a negative effect, however, with a lower slope, and the variable X

_{2}(docetaxel/lipids ratio) has a positive effect on particle size. Despite showing an opposed effect or additive impact, the results of the effects may not be statistically significant. In a previous study, when varying amplitude, cavitation cycle and time, a significant change was observed in the formation of unilamellar liposomes, and an amplitude of 40% was considered effective for obtaining nanometric sizes [32].

^{+}ions [45,53,54,55,56].

_{1}, Y

_{2}and Y

_{3}—encapsulation efficiency, particle size and PDI, respectively—were subject to multiple regression, generating a second order polynomial equation in a full quadratic model, in which the highest reliability coefficients (R

^{2}) were selected based on linear equations [57]. The coefficient values and equations obtained for the model are described in Table 3.

_{1}) and particle size (Y

_{2}) response model, as these presented the highest correlation coefficients (R

^{2}) [58].

_{1}) the isolated term X

_{1}and the interactions X

_{1}X

_{2}and X

_{1}X

_{3}favored the encapsulation efficiency response, although the isolated terms of X

_{2}and X

_{3}and their interaction (X

_{2}X

_{3}) had an antagonistic effect, negatively influencing the response. In the particle size response (Y

_{2}) the isolated terms DOPE/CHEMS molar ratio (X

_{1}) and sonication time (X

_{3}) and the interaction between them (X

_{1}X

_{3}) presented positive coefficients, denoting a favorable effect in obtaining the desired response. In the coefficients that presented the term X

_{2}(molar ratio DTX/lipids) and in the interactions X

_{1}X

_{2}and X

_{2}X

_{3}, the response was unfavorable due to negative coefficients, representing an antagonistic effect on these factors for the Y

_{2}response [57]. The term corresponding to the molar ratio of lipids had statistical significance for the dependent factor Y

_{1}(encapsulation efficiency). This result was expected, since the proportion of two lipid components is attributed as a critical point for the manufacture of liposomes with a potential to favor the Y

_{1}response [22,57]. The other terms showed little statistical significance.

_{3}) (Figure 3C) corresponds to the best attribution of homoscedasticity compared to the other responses [9,59]. The observation order vs. residual graphs, as well as the normal probability of residuals, showed acceptable residuals that supported the continuation of the analyses for formulation optimized by the statistical model [59].

_{1}, Y

_{2}and Y

_{3}were 73.7%, 115.7 nm and 0.297, respectively. The experimental values for optimized formulation were 88.65% of encapsulation efficiency (relative error = 20.3%), a particle size of 107.17 nm (relative error = 7.4%) and a polydispersity index equal to 0.213 (relative error 28.4%).

^{−1}. It is observed that the bands at 1165 cm

^{−1}e 1247 cm

^{−1}show the characteristic C-O stretching of ester [11]. The spectrum shows the peak referring to the N-H stretching of secondary amide (band in 3460 cm

^{−1}). It is also observed the strong stretching of C=O at 1701 cm

^{−1}, characteristic of amide. The spectrum also revealed bands at 1490 cm

^{−1}, 1452 cm

^{−1}consistent with the C=C aromatic ring. In the spectrum of the blank liposome, a characteristic main peak can be observed at 1740 cm

^{−1}(COOH vibration), as well as a wide O-H stretch between 3180–3500 cm

^{−1}. The peaks at 2853/2923 cm

^{−1}can be related to the symmetrical and asymmetrical CH2 stretching of the DSPE-PEG 2000 structure [64]. Weak absorption at 1645 cm

^{−1}indicates C=C stretching and the C-O-C stretching (1059 cm

^{−1}and 995 cm

^{−1}) belongs to the DSPE-PEG portion. The asymmetric stretching vibration of the P=O group can be identified by a peak at 1238 cm

^{−1}, which can be attributed to DSPE or DOPE, phospholipids. As for the liposome containing DTX (Figure 5B), the characteristic peaks of the drug (N-H stretch of the amide at 3460 cm

^{−1}and vibration of the ester bond around 1722 cm

^{−1}) are not in evidence, which could be explained by the fact that the drug is encapsulated in the matrix structure of the liposome, or due to the overlapping of the chemical bond bands of DTX and lipids [11]. Figure 5B(d,e) shows the FTIR spectra of the free cetuximab and blank immunoliposome, in order to evaluate the antibody functional groups and their presence in the immunoliposome structure after functionalization. Cetuximab exhibits a characteristic peak identified at 3270 cm

^{−1}in Figure 5B, which corresponds to the stretching vibration due to the O-H groups. Additionally, 2931 cm

^{−1}is the vibration attributed to the asymmetric stretching of C-H. Another characteristic peak is evidenced at 1696 cm

^{−1}, characteristic of the N-H group, and at 1712 cm

^{−1}, attributed to the C=O stretching of amide [65]. The characteristic peaks of the antibody were also found in the spectrum of the blank immunoliposome, signaling that cetuximab was attached to the nanoparticle with the appearance of the characteristic chemical groups. These peaks are more subtle, because the antibody is diluted in the formulation.

_{50}for DU145 (28.28 and 33.55 nM, respectively) when compared to PC3 (65.74 and 55.77 nM, respectively), thus being more cytotoxic for that cell line. Furthermore, in relation to the immunoliposome, as expected, the lowest IC

_{50}among all formulations was observed for DU145 (12.60 nM), demonstrating, when compared to the IC

_{50}of DTX solution (33.55 nM), that there was a greater targeting of the nanoparticle to this cell line, due to the higher expression of anti-EGFR receptors. Therefore, the immunoliposome increased cytotoxicity, with a decrease in IC

_{50}, which is equivalent to a value of 2.66 and 2.24 times lower than the DTX solution and the liposome, respectively, in DU145 cells.

## 4. Conclusions

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

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**Figure 1.**Response surface plots showing the effects of various formulation parameters on the responses encapsulation efficiency (Y

_{1}), particle size (Y

_{2}) and polydispersity index (Y

_{3}). The (

**A**–

**C**) response surface plots relate DOPE/CHEMS ratio (X

_{1}), docetaxel/lipid ratio (X

_{2}) and sonication time (X

_{3}) to encapsulation efficiency (Y

_{1}). The (

**D**–

**F**) plots to the effects of X

_{1}, X

_{2}and X

_{3}on particle size (Y

_{2}), and (

**G**–

**I**) to the effects of X

_{1}, X

_{2}and X

_{3}on the polydispersity index (Y

_{3}).

**Figure 2.**Perturbation plot and optimization graph. The perturbation plot (

**I**) shows the effects of the variables DOPE/CHEMS ratio (green), docetaxel/lipid ratio (blue) and sonication time (gray) for encapsulation efficiency (EE%). Graph (

**II**) shows the effects of the same variables on particle size (nm). Graph (

**III**) presents the graphical optimization for the desired formulation.

**Figure 3.**Graphs of normal probability (percentage vs. residual), of residuals versus adjusted values and observation order versus residual in relation to variables Y

_{1}, Y

_{2}and Y

_{3}. The graphs were combined and aligned in the figure in relation to each response analyzed in the experimental design, in which the line graphs (

**A**) relate to the encapsulation efficiency (Y

_{1}), in (

**B**) to the particle size (Y

_{2}) and in (

**C**) the polydispersity index (Y

_{3}).

**Figure 4.**Liposome elution profile with DIO fluorophore, free antibody, docetaxel immunoliposome with 2 mg antibody, docetaxel immunoliposome with 3 mg antibody and docetaxel immunoliposome with 3 mg antibody (Incubation 37

**°**C) by size exclusion chromatography on Sepharose CL-4B (

**A**) Graph of thermophoresis of cetuximab antibody, total immunoliposome, purified immunoliposome and liposome (

**B**) SDS-Page gel electrophoresis for free cetuximab and blank and docetaxel-containing immunoliposomes (

**C**).

**Figure 5.**Solid state characterization of lyophilized liposomes: (

**A**) powder X-ray: diffraction patterns of DTX (

**a**), unloaded liposome (

**b**), liposome DTX (

**c**), unloaded immunoliposome (

**d**), DTX immunoliposome (

**e**), PBS (

**f**) and sucrose (

**g**). (

**B**) FTIR: DTX (

**a**), unloaded liposome (

**b**), liposome DTX (

**c**), Cetuximab (

**d**), unloaded immunoliposome (

**e**). (

**C**) Differential scanning calorimetry: curves of DTX (

**a**), unloaded liposome (

**b**), DTX liposome (

**c**), unloaded immunoliposome (

**d**) and DTX immunoliposome (

**e**). (

**D**) Thermogravimetry: curve of DTX (

**a**), unloaded liposome (

**b**), DTX liposome (

**c**), unloaded immunoliposome (

**d**) and DTX immunoliposome (

**e**). The curves were vertically translated for clarity.

**Figure 6.**Image of the morphology of the liposome with drug (

**A**,

**B**), immunoliposome with drug (

**D**,

**E**), histograms of roughness of the liposome with drug (

**C**), histograms of the roughness of the immunoliposome with drug (

**F**), graph of mean roughness of liposome and immunoliposome DTX (

**G**). * p < 0.05.

**Figure 7.**Particle size distribution: DTX liposome by MFA (

**A**), DTX liposome by DLS (

**C**), DTX immunoliposome by MFA (

**B**), DTX immunoliposome by DLS (

**D**), in vitro release study of DTX-loaded liposome and DTX solution using dialysis membrane (

**E**).

**Figure 8.**Cell viability by the MTT assay at 72 h incubation. PC3 cell line (

**A**), DU145 cell line (

**B**).

**Figure 9.**Cellular internalization of the liposome and immunoliposome. PC3 cell line (

**A**), DU145 cell line (

**B**). Indirect ELISA to assess the binding ability of cetuximab, immunoliposome and liposome to the EGFR antigen (

**C**). Confocal microscopy (20× objective) of liposome in PC3 cells (

**D**), immunoliposome in PC3 cells (

**E**), liposome in DU145 cells (

**F**), immunoliposome in DU145 cells (

**G**). Two-way ANOVA test with Bonferroni posttest between samples. *** p < 0.01 (

**A**,

**B**). One-way ANOVA test with Tukey posttest between samples. **** p < 0.05 (

**C**).

Independent Variable | Level Used | |||
---|---|---|---|---|

Symbol | Low (−1) | Medium (0) | High (+1) | |

X_{1}: DOPE:CHEMS ratio | DP/CHEM | 1 | 1.5 | 2.5 |

X_{2}: Docetaxel:lipids ratio | DTX/LP | 1:15 | 1:20 | 1:30 |

X_{3}: Sonication time | ST | 2.5 min | 5 min | 10 min |

Fixed Component | DSPE-PEG 0.5 mol | |||

Response variables | Goal | |||

Y_{1}: Encapsulation efficiency (%) | EE (%) | Maximum (100%) | ||

Y_{2}: Particle size (nm) | Size (nm) | Optimum (<200 nm) | ||

Y_{3}: Polydispersity index | PDI | Minimize |

**Table 2.**Box–Behnken design representing experimental runs with independent variables at three levels (−1, 0, 1) and observed responses (EE%, Size and PDI).

Formulation Run | X_{1} (DP/CHEM) | X_{2} (DTX/LP) | X_{3} (ST) | Y_{1} [EE (%)] | Y_{2} [Size (nm)] | Y_{3} (PDI) | |||
---|---|---|---|---|---|---|---|---|---|

Level | Tested Ratio | Level | Tested Ratio | Level | Time Tested | ||||

1 | −1 | 1.0 | −1 | 1:15 | 0 | 5 | 32.4 ± 2.28 | 121.6 ± 2.2 | 0.350 ± 0.019 |

2 | 1 | 2.5 | −1 | 1:15 | 0 | 5 | 22.88 ± 1.12 | 213.3 ± 7.3 | 0.394 ± 0.032 |

3 | −1 | 1.0 | 1 | 1:30 | 0 | 5 | 23.88 ± 4.89 | 292.9 ± 21.9 | 0.361 ± 0.044 |

4 | 1 | 2.5 | 1 | 1:30 | 0 | 5 | 25.15 ± 6.00 | 163.1 ± 0.8 | 0.355 ± 0.004 |

5 | −1 | 1.0 | 0 | 1:20 | −1 | 2.5 | 21.42 ± 2.08 | 182.5 ± 1.8 | 0.416 ± 0.005 |

6 | 1 | 2.5 | 0 | 1:20 | −1 | 2.5 | 31.73 ± 3.21 | 126.8 ± 3.1 | 0.386 ± 0.037 |

7 | −1 | 1.0 | 0 | 1:20 | 1 | 10 | 16.36 ± 3.70 | 130.9 ± 4.3 | 0.259 ± 0.001 |

8 | 1 | 2.5 | 0 | 1:20 | 1 | 10 | 13.45 ± 0.57 | 156 ± 0.4 | 0.303 ± 0.018 |

9 | 0 | 1.5 | −1 | 1:15 | −1 | 2.5 | 89.36 ± 8.77 | 145.1 ± 0.7 | 0.200 ± 0.004 |

10 | 0 | 1.5 | 1 | 1:30 | −1 | 2.5 | 88.97 ± 13.78 | 114.1 ± 0.6 | 0.253 ± 0.009 |

11 | 0 | 1.5 | −1 | 1:15 | 1 | 10 | 33.26 ± 9.17 | 228.8 ± 6.2 | 0.481 ± 0.047 |

12 | 0 | 1.5 | 1 | 1:30 | 1 | 10 | 28.01 ± 6.43 | 137.7 ± 1.2 | 0.400 ± 0.022 |

13 | 0 | 1.5 | 0 | 1:20 | 0 | 5 | 32.72 ± 6.85 | 144.0 ± 6.4 | 0.480 ± 0.013 |

14 | 0 | 1.5 | 0 | 1:20 | 0 | 5 | 43.17 ± 5.24 | 193.9 ± 27.2 | 0.453 ± 0.043 |

15 | 0 | 1.5 | 0 | 1:20 | 0 | 5 | 19.31 ± 0.74 | 137.1 ± 1.9 | 0.387 ± 0.198 |

**Table 3.**Regression analysis for the encapsulation efficiency (Y

_{1}), particle size (Y

_{2}) and polydispersity index (Y

_{3}) using an interaction model based on the effect of the DOPE:CHEMS ratio (X

_{1}), docetaxel:lipids ratio (X

_{2}) and sonication time (X

_{3}).

Y_{1} [EE (%)] | Y_{2} [Size (nm)] | Y_{3} (PDI) | ||||
---|---|---|---|---|---|---|

Coeff. | p-Value | Coeff. | p-Value | Coeff. | p-Value | |

Intercept | 139.426 | 0.345 | 108.439 | 0.822 | −0.545 | 0.580 |

X_{1} | 155.460 | 0.094 | 2.211 | 0.993 | 0.331 | 0.553 |

X_{2} | −15.354 | 0.138 | −5.294 | 0.866 | 0.039 | 0.545 |

X_{3} | −19.119 | 0.178 | 41.911 | 0.362 | 0.089 | 0.336 |

X_{1}^{2} | −47.378 | 0.051 | 36.928 | 0.587 | −0.076 | 0.581 |

X_{2}^{2} | 0.346 | 0.109 | 0.480 | 0.466 | −0.001 | 0.549 |

X_{3}^{2} | 1.388 | 0.109 | −2.482 | 0.355 | −0.006 | 0.284 |

X_{1}X_{2} | 0.304 | 0.843 | −7.727 | 0.182 | −0.002 | 0.847 |

X_{1}X_{3} | 0.289 | 0.925 | 4.016 | 0.704 | −0.003 | 0.882 |

X_{2}X_{3} | −0.159 | 0.597 | −0.684 | 0.509 | 0.000 | 0.917 |

R^{2} | 81.81(%) | 51.42(%) | 30.66(%) | |||

Regression equation of the fitted model | ||||||

${\mathrm{Y}}_{1}=139.426+155.460{\mathrm{X}}_{1}-15.345{\mathrm{X}}_{2}-19.119{\mathrm{X}}_{3}+0.304{\mathrm{X}}_{1}{\mathrm{X}}_{2}+0.289{\mathrm{X}}_{1}{\mathrm{X}}_{3}-0.159{\mathrm{X}}_{2}{\mathrm{X}}_{3}-47.378{\mathrm{X}}_{1}^{2}+0.346{\mathrm{X}}_{2}^{2}+1.388{\mathrm{X}}_{3}^{3}$ | ||||||

${\mathrm{Y}}_{2}=108.439+2.211{\mathrm{X}}_{1}-5.294{\mathrm{X}}_{2}+41.911{\mathrm{X}}_{3}-7.727{\mathrm{X}}_{1}{\mathrm{X}}_{2}+4.016{\mathrm{X}}_{1}{\mathrm{X}}_{3}-0.684{\mathrm{X}}_{2}{\mathrm{X}}_{3}+36.928{\mathrm{X}}_{1}^{2}+0.480{\mathrm{X}}_{2}^{2}-2.482{\mathrm{X}}_{3}^{3}$ | ||||||

${\mathrm{Y}}_{3}=-0.545586+0.331{\mathrm{X}}_{1}+0.039{\mathrm{X}}_{2}+0.089{\mathrm{X}}_{3}-0.002{\mathrm{X}}_{1}{\mathrm{X}}_{2}-0.003{\mathrm{X}}_{1}{\mathrm{X}}_{3}-0.0002{\mathrm{X}}_{2}{\mathrm{X}}_{3}-0.076{\mathrm{X}}_{1}^{2}-0.001{\mathrm{X}}_{2}^{2}-0.0059{\mathrm{X}}_{3}^{3}$ |

_{1}X

_{2}, X

_{1}X

_{3}and X

_{2}X

_{3}) and the quadratic relationships are represented by higher order terms (i.e., X

_{1}

^{2}, X

_{2}

^{2}and X

_{3}

^{2}). R

^{2}represents the coefficient of determination.

**Table 4.**Physicochemical characterization of immunoliposome formulations prepared by direct conjugation.

Immunoliposome with 2 mg Antibody | |||

Blank Immunoliposome * | DTX Immunoliposome * | ||

Size (nm) | 107.4 ± 2.05 | 106.8 ± 4.45 | |

PDI | 0.224 ± 0.013 | 0.224 ± 0.013 | |

Zeta (mV) | −16.7 ± 1.42 | -17.1 ± 1.50 | |

EC% | 9.84% | 10.68% | |

EE% | - | 85.84 ± 3.70 | |

Immunoliposome with 3 mg Antibody | |||

Blank immunoliposome * | DTX immunoliposome * | DTX immunoliposome at 37 °C | |

Size (nm) | 111.07 ± 0.51 | 111.47 ± 0.49 | 156.77 ± 1.67 |

PDI | 0.197 ± 0.01 | 0.245 ± 0.01 | 0.245 ± 0.00 |

Zeta (mV) | −17.77 ± 1.20 | −17.77 ± 1.20 | −17.67 ± 1.35 |

EC% | 11.65% | 6.91% | 14.06% |

EE% | - | 75.8 ± 15.30 | 86.0 ± 14.29 |

**Table 5.**IC

_{50}of the formulations tested in the two cell lines (PC3 and DU145) with their respective confidence interval.

PC3 | ||

Formulation | IC_{50} (nM) | Confidence interval |

DTX solution | 55.77 ± 9.21 | 38.76 to 81.80 |

DTX liposome | 65.74 ± 14.61 | 41.17 to 110.3 |

DTX immunoliposome | 152.1 ± 25.43 | 106.2 to 225.5 |

Cetuximab | - | - |

DU145 | ||

Formulation | IC_{50} (nM) | Confidence interval |

DTX solution | 33.55 ± 7.20 | 20.91 to 55.33 |

DTX liposome | 28.28 ± 4.60 | 19.80 to 40.99 |

DTX immunoliposome | 12.60 ± 2.50 | 8.128 to 19.91 |

Cetuximab | - | - |

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**MDPI and ACS Style**

Moreira, T.d.S.; Silva, A.D.O.; Vasconcelos, B.R.F.; Santos, E.d.S.; de Sousa, A.C.C.; de Freitas, J.V.B.; de Oliveira, Y.S.; Vidal, L.M.T.; Ribeiro, F.d.O.S.; de Araújo, A.R.;
et al. DOPE/CHEMS-Based EGFR-Targeted Immunoliposomes for Docetaxel Delivery: Formulation Development, Physicochemical Characterization and Biological Evaluation on Prostate Cancer Cells. *Pharmaceutics* **2023**, *15*, 915.
https://doi.org/10.3390/pharmaceutics15030915

**AMA Style**

Moreira TdS, Silva ADO, Vasconcelos BRF, Santos EdS, de Sousa ACC, de Freitas JVB, de Oliveira YS, Vidal LMT, Ribeiro FdOS, de Araújo AR,
et al. DOPE/CHEMS-Based EGFR-Targeted Immunoliposomes for Docetaxel Delivery: Formulation Development, Physicochemical Characterization and Biological Evaluation on Prostate Cancer Cells. *Pharmaceutics*. 2023; 15(3):915.
https://doi.org/10.3390/pharmaceutics15030915

**Chicago/Turabian Style**

Moreira, Thais da Silva, Alan Denis Olivindo Silva, Bianca Rodrigues Farias Vasconcelos, Elias da Silva Santos, Ana Carolina Cruz de Sousa, João Vito Barroso de Freitas, Yara Santiago de Oliveira, Laura Maria Teodorio Vidal, Fábio de Oliveira Silva Ribeiro, Alyne Rodrigues de Araújo,
and et al. 2023. "DOPE/CHEMS-Based EGFR-Targeted Immunoliposomes for Docetaxel Delivery: Formulation Development, Physicochemical Characterization and Biological Evaluation on Prostate Cancer Cells" *Pharmaceutics* 15, no. 3: 915.
https://doi.org/10.3390/pharmaceutics15030915