A Screening Study for the Development of Simvastatin-Doxorubicin Liposomes, a Co-Formulation with Future Perspectives in Colon Cancer Therapy
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Methods
2.2.1. Implementation of the QbD Concept
2.2.2. Liposomes Preparation
2.2.3. Characterization of Liposomes
Determination of SIM and DOX Entrapped Concentration
Determination of Liposomal Size and PdI
Determination of Liposomal Morphology using Transmission Electron Microscopy (TEM) Analysis
In Vitro Release Study
2.2.4. Cell Co-Culture
2.2.5. Cell Proliferation Assay
3. Results and Discussions
3.1. Setting the QTPP and the CQAs
3.2. Risk Analysis
3.3. DoE
3.4. DoE Analysis
3.4.1. The Influence of the Formulation Factors on Drug Entrapped Concentration
3.4.2. The Influence of the Formulation Factors on EE%
3.4.3. The Influence of the Formulation Factors on Liposomal Size
3.4.4. The Influence of the Formulation Factors on PdI
3.4.5. The Influence of the Formulation Factors on Zeta Potential
3.5. TEM Analysis
3.6. In Vitro Release Study
3.7. The Effects of SIM and DOX on the Proliferation of C26 Murine Colon Carcinoma Cells Co-Cultured with Murine Macrophages
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
References
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PL (mM) | 44 | 77 |
---|---|---|
DOX (µg/mL) | 176.86 ± 25.39 | 194.9 ± 66.43 |
SIM (µg/mL) | 1267.42 ± 121.96 | 1552.44 ± 76.13 |
Quality Target Product Profile | Target | Justification | |
---|---|---|---|
Formulation | Liposomes | - | To deliver simultaneously SIM and DOX at the tumor site |
Route of administration | Intravenous | - | To increase the bioavailability of both active substances at the tumor site |
Quality attributes | Pegylated liposomes | - | To ensure prolonged blood circulation time for liposomes |
Zeta potential | ≤−30 mV | Values higher than +30 mV or lower than −30 mV are required for a good colloidal stability of the formulation | |
EE% | 50–100% | Reduced production losses | |
Drug entrapped concentrations | DOX > 120 µg/mL | Increased dosing intervals | |
SIM > 1200 µg/mL | |||
SIM to DOX molar ratio | 12 to 1 | Securing the right ratio between the two active substances in liposomal formulation is essential to achieve the desired cytotoxic effect at the tumor site | |
Spherical shape | - | Active loading of DOX into liposomes may lead to a deformation of liposomes shape and membrane disruption | |
Size | 50–200 nm | Enhanced permeability retention effect is dependent on the liposomal size, values smaller than 200 nm being indicated; Liposomes smaller than 50 nm are quickly eliminated by the spleen and through kidney filtration | |
PdI | <0.2 | A monodisperse liposomal suspension is critical for predictable in vivo behavior | |
Prolonged in vitrorelease profile | - | Required in order to increase the dosing interval and to prevent off-site drug release |
Parameter | Failure Mode | Failure Effects | Potential Causes | Control Methods | S | D | O | RPN |
---|---|---|---|---|---|---|---|---|
Drug entrapped concentration and EE% | -Reduced drug entrapped concentration; -Reduced EE% | -Drug losses; -Increased production costs; -Frequent dosing required; -Enhanced administered doses; -Reduced patient compliance; -Diminished therapeutic effect | PL physiochemical properties | Use of PL with an increased Tm | 3 | 1 | 5 | 15 |
PL concentration | Identification of an appropriate concentration range | 5 | 4 | 5 | 100 | |||
SIM concentration | 5 | 4 | 5 | 100 | ||||
DOX concentration | 5 | 4 | 5 | 100 | ||||
PL to cholesterol molar ratio | 5 | 1 | 1 | 5 | ||||
DOX loading method | Use of active loading method | 5 | 1 | 1 | 5 | |||
Type of sulphate salt | Use of AS salt | 5 | 1 | 3 | 15 | |||
Sulphate salt concentration | Use of a 250 mM salt concentration | 3 | 1 | 3 | 9 | |||
The pH of the hydration medium | Optimization of the pH of the AS solution | 3 | 4 | 5 | 60 | |||
Establishing a pH gradient | Optimization of liposomes purification step | 1 | 1 | 1 | 1 | |||
DOX to PL molar ratio | Use of a DOX to PL molar ratio smaller than 0.95 | 4 | 1 | 2 | 8 | |||
Incubation time | Optimization of incubation time of SIM-LCL with DOX | 5 | 4 | 5 | 100 | |||
Incubation temperature | Performing the incubation step at 60 °C | 4 | 1 | 1 | 4 | |||
Size and PdI | -Multilamellar vesicles; -Polydisperse suspension; -Increased liposomal size | -Reduced internal liposomal volume for DOX encapsulation; -Diminished therapeutic effect; -Fast elimination through reticuloendothelial system; -Reduced colloidal stability | Size reduction method | Application of extrusion process | 1 | 1 | 1 | 1 |
Optimizing the number of extrusion cycles | 5 | 1 | 3 | 15 | ||||
Use of membranes with a reduced pore size | 4 | 1 | 4 | 16 | ||||
Prolonged blood circulation time | Rapid removal from blood circulation | -Frequent dosing required; -Diminished therapeutic effect | Formulation of conventional liposomes | Use of pegylated PL | 5 | 1 | 4 | 20 |
Prolonged release profile | Fast drug release rate | -Diminished therapeutic effect; -Frequent dosing required; -Off-site drug release; -Increased number of side effects; -Reduced patient compliance; -Increased health care costs | Use of reduced PL concentration | Optimization of PL concentration | 4 | 4 | 5 | 80 |
SIM and DOX concentration | Optimization of drug concentration | 2 | 2 | 5 | 20 | |||
Use of PL with a reduced Tm | Use of PL with an increased Tm | 3 | 1 | 4 | 12 | |||
Zeta potential | Values in the range of −20 mV to +20 mV | -Reduced colloidal stability; -Interactions with blood constituents and rapid removal from blood circulation; -Vesicle agglomeration; -Sedimentation | PL concentration | Optimization of PL concentration | 5 | 4 | 5 | 100 |
Ionic strength of the liposomes external medium; | Use of NaCl for dialysis | 3 | 2 | 2 | 12 | |||
Type of PL | Use of charged PL | 3 | 3 | 2 | 18 |
Exp Name | X1 | X2 | X3 | X4 | X5 | Y1 | Y2 | Y3 | Y4 | Y5 | Y6 | Y7 | Y8 | Y9 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
N1 | 11 | 2 | 0.25 | 5.00 | 15 | 157.67 | 18.77 | 59.64 | 28.67 | 118.46 | 0.023 | 110.33 | 0.028 | −22.56 |
N2 | 11 | 7 | 0.50 | 5.00 | 30 | 297.51 | 10.20 | 123.95 | 42.59 | 111.86 | 0.060 | 106.76 | 0.091 | −20.70 |
N3 | 11 | 12 | 0.75 | 5.00 | 45 | 126.60 | 2.52 | 173.84 | 38.14 | 108.86 | 0.047 | 105.83 | 0.066 | −25.16 |
N4 | 44 | 2 | 0.25 | 5.00 | 30 | 364.58 | 44.05 | 156.66 | 91.45 | 123.33 | 0.014 | 122.00 | 0.036 | −34.66 |
N5 | 44 | 7 | 0.50 | 5.00 | 45 | 1129.08 | 38.66 | 325.19 | 84.20 | 118.06 | 0.030 | 110.16 | 0.056 | −31.20 |
N6 | 44 | 12 | 0.75 | 5.00 | 15 | 1809.98 | 35.80 | 378.42 | 75.07 | 117.93 | 0.025 | 115.16 | 0.051 | −36.46 |
N7 | 77 | 2 | 0.50 | 5.00 | 15 | 224.54 | 27.03 | 285.83 | 74.00 | 132.93 | 0.050 | 125.53 | 0.039 | −35.53 |
N8 | 77 | 7 | 0.75 | 5.00 | 30 | 1130.26 | 38.61 | 439.40 | 87.16 | 122.50 | 0.031 | 117.10 | 0.060 | −34.46 |
N9 | 77 | 12 | 0.25 | 5.00 | 45 | 1384.17 | 27.61 | 202.12 | 97.16 | 125.16 | 0.020 | 116.80 | 0.034 | −37.00 |
N10 | 11 | 2 | 0.75 | 5.50 | 45 | 232.02 | 25.66 | 189.37 | 41.55 | 117.26 | 0.046 | 115.30 | 0.075 | −21.13 |
N11 | 11 | 7 | 0.25 | 5.50 | 15 | 185.80 | 6.31 | 61.30 | 29.47 | 114.56 | 0.040 | 110.36 | 0.079 | −37.93 |
N12 | 11 | 12 | 0.50 | 5.50 | 30 | 238.31 | 4.74 | 167.75 | 57.64 | 116.03 | 0.057 | 110.70 | 0.068 | −28.50 |
N13 | 44 | 2 | 0.50 | 5.50 | 45 | 336.65 | 39.76 | 275.85 | 88.19 | 128.20 | 0.065 | 126.00 | 0.056 | −30.93 |
N14 | 44 | 7 | 0.75 | 5.50 | 15 | 1335.59 | 45.73 | 410.57 | 88.31 | 120.60 | 0.046 | 115.33 | 0.062 | −25.70 |
N15 | 44 | 12 | 0.25 | 5.50 | 30 | 1200.86 | 23.97 | 66.84 | 39.88 | 116.86 | 0.045 | 109.43 | 0.059 | −27.60 |
N16 | 77 | 2 | 0.75 | 5.50 | 30 | 244.61 | 29.22 | 401.70 | 79.68 | 135.76 | 0.029 | 132.36 | 0.040 | −31.96 |
N17 | 77 | 7 | 0.25 | 5.50 | 45 | 1130.51 | 37.73 | 111.75 | 53.72 | 121.06 | 0.045 | 116.03 | 0.052 | −35.53 |
N18 | 77 | 12 | 0.50 | 5.50 | 15 | 1361.95 | 27.09 | 251.59 | 65.14 | 120.53 | 0.052 | 115.50 | 0.022 | −33.34 |
N19 | 44 | 7 | 0.50 | 5.00 | 15 | 1969.29 | 67.21 | 228.17 | 78.40 | 120.86 | 0.028 | 114.36 | 0.054 | −29.03 |
N20 | 44 | 7 | 0.50 | 5.00 | 15 | 2066.85 | 70.71 | 274.19 | 94.21 | 120.43 | 0.021 | 115.76 | 0.050 | −34.63 |
N21 | 44 | 7 | 0.50 | 5.00 | 15 | 1392.23 | 47.50 | 253.12 | 86.97 | 119.83 | 0.045 | 115.40 | 0.045 | −26.70 |
Liposomal Formulation | X1 | X2 | X3 | Y1 | Y2 | Y3 | Y4 | Y7 | Y8 | Y9 |
---|---|---|---|---|---|---|---|---|---|---|
DOX-LCL | 44 | - | 0.5 | - | - | 165.47 | 56.08 | 129.4 | 0.055 | −28.7 |
SIM1-DOX-LCL | 44 | 2 | 0.25 | 260.74 | 35.59 | 89.90 | 62.33 | 119.8 | 0.050 | −28.3 |
SIM2-DOX-LCL | 44 | 12 | 0.5 | 998.49 | 19.92 | 115.37 | 37.27 | 104.3 | 0.071 | −27 |
Pharmaceutical Formulation | Tested Drug Concentration | IC50 (μM) |
---|---|---|
SIM solution | 0.041–20.7 μM | 3 ± 0.560 |
DOX solution | 0.007–10 μM | 0.25 ± 0.047 |
SIM1 + DOX (variable) | 0.934 μM SIM + (0.015–8 μM) DOX | 0.06 ± 0.036 |
SIM2 + DOX (variable) | 3 μM SIM+ (0.019–8 μM) DOX | 0.27 ± 0.114 |
DOX-LCL | 0.007–5 μM DOX | 0.27 ± 0.127 |
SIM1-DOX-LCL | 0.934 μM SIM + (0.007–5) μM DOX | 0.37 ± 0.086 |
SIM2-DOX-LCL | 3 μM SIM + (0.007–5) μM DOX | 0.08 ± 0.044 |
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Barbălată, C.I.; Porfire, A.S.; Sesarman, A.; Rauca, V.-F.; Banciu, M.; Muntean, D.; Știufiuc, R.; Moldovan, A.; Moldovan, C.; Tomuță, I. A Screening Study for the Development of Simvastatin-Doxorubicin Liposomes, a Co-Formulation with Future Perspectives in Colon Cancer Therapy. Pharmaceutics 2021, 13, 1526. https://doi.org/10.3390/pharmaceutics13101526
Barbălată CI, Porfire AS, Sesarman A, Rauca V-F, Banciu M, Muntean D, Știufiuc R, Moldovan A, Moldovan C, Tomuță I. A Screening Study for the Development of Simvastatin-Doxorubicin Liposomes, a Co-Formulation with Future Perspectives in Colon Cancer Therapy. Pharmaceutics. 2021; 13(10):1526. https://doi.org/10.3390/pharmaceutics13101526
Chicago/Turabian StyleBarbălată, Cristina Ioana, Alina Silvia Porfire, Alina Sesarman, Valentin-Florian Rauca, Manuela Banciu, Dana Muntean, Rareș Știufiuc, Alin Moldovan, Cristian Moldovan, and Ioan Tomuță. 2021. "A Screening Study for the Development of Simvastatin-Doxorubicin Liposomes, a Co-Formulation with Future Perspectives in Colon Cancer Therapy" Pharmaceutics 13, no. 10: 1526. https://doi.org/10.3390/pharmaceutics13101526
APA StyleBarbălată, C. I., Porfire, A. S., Sesarman, A., Rauca, V.-F., Banciu, M., Muntean, D., Știufiuc, R., Moldovan, A., Moldovan, C., & Tomuță, I. (2021). A Screening Study for the Development of Simvastatin-Doxorubicin Liposomes, a Co-Formulation with Future Perspectives in Colon Cancer Therapy. Pharmaceutics, 13(10), 1526. https://doi.org/10.3390/pharmaceutics13101526