Picto La Vague

January 2024

La Vague n°80

Microbiology

Download the french issue

Sommaire

✻

Design of smart galenic models (stimuli-sensitive) for the formulation of active molecules

Among the different sustained release systems for active pharmaceutical ingredients (API), stimuli-sensitive polymers, called “smart” materials, have gained interest in galenic development given the change in the configuration of the polymer network according to the chosen parameter of its environment (pH, temperature, redox, polarity, etc.). This article describes the methodology for developing an extended release formulation based on Poly(N- isopropylacrylamide ) (PNIPAm), a biocompatible, thermosensitive polymer with unique and versatile properties, the physicochemical characterization of aging, as well as its encapsulation capacity

Conception Modèles Galéniques Intelligents Formulation Molécules Actives La Vague 2024 80 Fig1

With the increasing prevalence of chronic diseases, growth in the biologics market, increasing investments in R&D and technological advancements accompanied by new product launches, forecasting the average annual growth rate of the global biologics market would be 5.9% [1]. Several approaches to drug delivery through modifications (of the drug, its environment and delivery systems) have been developed to improve the effectiveness and release of API while minimizing its accumulation outside target [2]. Among the different drug delivery systems (Drug Delivery Systems, DDS), several families of polymers have been described [3] and divided into two families: natural and synthetic polymers (Figure 2) including biodegradable and bioabsorbable polymers such as poly-lactic acid (polylactic acid, PLA) and poly-lactic -co -glycolic acid (poly- lactic – co-glycolic acid, PLGA).

Taking into account the 5 different generations of API, formulations using polymers are described:

1) small molecules (< 900 daltons): Naloxegol (Movantik®), PEGylated naloxone
2) proteins & peptides: Bovine Pegademase (Adagen®) , PEGylated protein ,
3) antibody: Certolizumab pegol (Cimzia®),
4) nucleic acids: Givosiran ( Givlaari®), an N- acetylgalactosamine -siRNA conjugate , ( GalNAc –siRNA),

5)cell therapy: CAR-T cells , (Chimeric Antigen receptor (CAR) inserted into the T cells), hydrogels based on Gelatin Methacryloyl (GelMA) recently developed since the marketing of Tisagenlecleucel ( Kymriah®) [4].

PNIPAm is a model polymer for its thermoresponsive properties as a smart polymer. It has unique properties in aqueous solution which are represented by both its phase diagram and Lower Critical Solution Temperature (LCST) described since 1968 by Heskins and Guillet [5] (Figure 3). Above this critical temperature, the PNIPAm polymer chains transition to a completely hydrophobic configuration. This causes the polymer chains to retract in the form of a globule and two phases appear [6] [7]. This property is even more interesting being close to that of the human body (LCST = 32°C) [8].

The work presented in this article shows the first phase of the PhD project, conducted within two laboratories EBI and ENSAM. One of the purposes of the research topic consists in studying the thermosensitive behavior of a model polymer, PNIPAm at its linear form. Besides, to verify the stability of this material over time, accelerated thermo-oxidative aging was carried out. Then in a second step, a PNIPAm gel is synthesized, according to a free radical polymerization, and its capacity to encapsulate an API is studied, supplemented with physicochemical characterizations.

1. Materials and methods

1.1 Materials

PNIPAm.  The linear polymer model used in this study is a Poly(N- isopropylacrylamide ) (CAS: 25189-55-3, Sigma-Aldrich) having a molar mass (supplier data) in number M n = 40,000 g.mol -1.

PNIPAm hydrogel.The synthesis of the PNIPAm hydrogel was adapted according to the protocol described by Lapointe [9]. The monomer, N- isopropylacrylamide (NIPAm) (CAS: 2210-25-5, Thermo Scientific) is recrystallized from diethyl ether (CAS: 60-29-7, VWR Chemicals). Then a radical polymerization in an aqueous medium is carried out. To summarize, 0.8 g of NIPAm, the monomer, 0.012 g of N, N’- methylenebisacrylamide (BIS), the cross-linking agent (CAS: 110-26-9, Sigma-Aldrich) and 0.0085 g of tetramethylethylenediamine (TEMED), acting as a reducing agent, accelerator of the redox reaction, (CAS: 110-18-9, VWR Chemicals) are homogenized in 10 mL of deionized water (DIW). Then, 340 µL of an ammonium persulfate (APS) solution (CAS: 7727-54-0, ACROS ORGANICS) (10% wt) is added to initiate the redox reaction [9]. The solutions are initially placed under a nitrogen flow for 1 hour and the synthesis is sealed for 24 hours at room temperature (20°C). Following the synthesis, a 7-day cleaning immersion in DIW is carried out.

1.2. Characterization of polymer stability

To study the thermosensitive behavior of linear PNIPAm and its stability, a method based on ultraviolet-visible (UV-Vis) spectroscopy measurements is applied. The approach consists of resolubilizing the polymer in DIW, before and after aging at 120°C (in a ventilated oven). Each sample was analyzed by UV-Vis at a wavelength of 651 nm with a heating ramp of 0.3°C.min -1 between 20°C and 40°C to characterize the phase transition temperature. To study the chemical stability of the chemical functions of the polymer during thermal aging, an analysis by Fourier Transform infrared spectroscopy (FTIR) by Attenuated Total Reflectance (ATR) is carried out in transmission mode. The FTIR analysis is carried out between wavenumbers ranging from 650 to 4000 cm -1, with a resolution of 4 cm -1, and a total of 32 recordings.

To study the variations in molar masses during thermal aging, analyzes by gel permeation chromatography (GPC) are carried out. The separation columns used T3000 and T6000 make it possible to analyze a range of 10,000 to 20,000,000 g.mol -1 and using tetrahydrofuran (THF) as mobile phase. The calibration curve is carried out using 5 standard samples of poly (methyl methacrylate) (PMMA) (M w = 4.7.10 3 –137.0.10 3 g.mol -1). The polymer powders (2.0 mg.mL -1) are analyzed at 35°C with a mobile phase flow rate of 1 mL.min -1.

1.3. Characterization of hydrogel synthesis and adsorption and desorption capacity

To verify the conformity of the synthesized polymer, one of the characterizations consists of an analysis by infrared spectroscopy (IRTF-ATR). To study the ability of the PNIPAm gel to encapsulate the API, the following protocol was implemented: Aqueous solutions of metronidazole (MTZ), at different concentrations (0.01, 0.5 and 2 mg. mL -1) are prepared. Then 0.04 g of dried gel is deposited in 20 mL of the solution at 20°C (made in triplicate). The absorbance is monitored by measuring λ max = 317 nm of the MTZ in the EDI by UV-Vis Spectroscopy for different time intervals (30min, 1h, 2h, 3h, 5h and 24h). A calibration curve is first created from MTZ solutions with concentrations ranging from 2 µg. mL -1 to 10 µg. mL -1. The saturation of the UV measurement appears for 25 µg. mL -1. Therefore, to monitor encapsulation, it is necessary to position yourself below this concentration. The calculation of the quantity of MTZ adsorbed per mass of PNIPAm is obtained according to equation (1).

With C 0 (mg. mL -1) the initial concentration, C t (mg. mL -1) the concentration at time t , m the mass of the support (g) and V the volume of solution (mL).

The calculation of the percentage of MTZ adsorbed is obtained according to equation (2):

% of MTZ adsorbed = x 100

2. Results

2.1 Thermosensitive properties and stability of the polymeric support

The linear PNIPAm studied has a phase diagram represented by the squares (Figure 4a). Below the turbidity temperatures (Tc), the PNIPAm solutions are homogeneous, and a single phase is observed. The amide functions, present on the side chain, are linked by hydrogen bonds to water, making the polymer completely soluble. When the temperature is increased beyond the Tc, the breakdown of the hydrogen bonds causes a rearrangement of the polymer chains and a polymer-polymer interaction is generated. A retraction is observed in the form of a globule and then a phase unmixing (Figure 3). The separation criterion here corresponds to a loss of 50% in transmittance. For solutions with a very low concentration of PNIPAm, the Tc are high. The latter, however, decrease very quickly when the PNIPAm content increases, until reaching a plateau around 25°C corresponding to the LCST at 50% by weight of PNIPAm. [8].

Following thermal aging of linear PNIPAm, an increase in Tc  is observed for the same concentration. For example, for a 10% solution by weight, a difference of 2.8°C is noted after 90 days of aging at a temperature of 120°C (Figure 4b). The more severe the aging, whether in terms of temperature or duration, the more significant the difference in Tc. Such a variation most certainly comes from a modification at the level of the polymer chains which interact with water differently once thermal aging has taken place. Structural characterizations are thus carried out to better understand the origin of this modification of behavior.

The typical infrared spectrum of PNIPAm is depicted in Figure 5a. The band at 3289 cm -1 corresponds to -OH bonds. The bands at 2973, 2928 and 2872 cm -1 correspond to the -CH3, -CH2 and -CH2 bonds of the isopropyl group as well as the bands at 1460 and 1386 cm -1. The amide group corresponds to the bands at 1633 cm -1 of the -C=O amide I bond and at 1537 cm -1 of the -C=O amide II bond. Finally, the band at 1367 cm -1 is specific to the primary chain of the carbon skeleton of PNIPAm. After thermal aging for 90 days at 120°C, no chemical modification is observed on the spectrum. These results would indicate that the chemical structure of the polymer remains stable and would not be responsible for the increase in Tc.

Analysis by GPC (Figure 5b) makes it possible to estimate the average molar mass of the aged/without aging polymer. After thermal aging, a polymer retention peak is slightly shifted towards a longer retention time. This demonstrates that the size of the polymer chains becomes shorter, following thermal aging. This shortening can be explained by chain cutting phenomena under the effect of thermo-oxidative aging. The size of the PNIPAm polymer chains strongly impacts the Tc as described by Furyk et al. [7]. The shorter the molar masses of PNIPAm, the higher the Tc, this is consistent with the results obtained.

2.2 Initial characterization of polymer gels

Following the polymerization and washing, drying in an oven at 40°C is carried out and the conformity of the hydrogel is analyzed (Figure 6). The IR spectrum of the synthesized gel shows the same structure as the commercial linear PNIPAm. The bonds corresponding to the amide, isopropyl groups and the carbon chain are clearly present.

3. MTZ adsorption/desorption study

After drying, aerogels are obtained and the adsorption of MTZ could be carried out by bringing the support into contact with an MTZ solution. A maximum adsorption of 40% with 468 mg.g -1 of MTZ/g of dry gel is obtained for an initial concentration of 2 mg. mL -1 and a contact time of 3 hours. After processing the results, several phenomena can be noted. First, the greater the initial concentration of MTZ, the greater the quantity adsorbed by the support. The second observation is that the equilibrium state reached after 24 hours does not offer the most optimal adsorption which returns to 0%. After 3 hours of contact, desorption into MTZ appears.

The desorption phase is also noted for other active molecules and appears between 1 and 3 hours of contact. The interactions between the DIW and the gel, which is rather hydrophilic, are very favorable, to the detriment of the API-support interactions, hence the importance of the choice of solvent during adsorption. The most suitable solvent for the API-PNIPAm couple is selected. Furthermore, other observations were made depending on the number of H-donor groups in the API, its logarithm of the octanol/water partition coefficient (logP) and its molecular weight. Finally, the evaluations in terms of stability and toxicity will be carried out according to the International Conference on Harmonization (ICH) Q8 (R2) guide[10].

3. Conclusion

The thermochemical stability of PNIPAm was studied. The accelerated thermo-oxidative aging carried out does not cause any chemical modification and imposes a slow chain cutting mechanism. The main consequence is a slight modification of the thermo-sensitive behavior. This polymer is therefore chemically stable to be applied in the pharmaceutical formulation. Furthermore, absorption is carried out on a network of PNIPAm in the form of a hydrogel, or reticulated form which is even more stable than the linear model. Consequently, these results confirm that the thermosensitive property will be maintained stable for at least 12 months at 120°C and thus for a longer period at room temperature, depending on the formulation and storage conditions of the material. Contact with an antibiotic, MTZ, was carried out and it easily integrated the PNIPAm network but did not bind to it, the water molecules having a greater affinity with the PNIPAm gel. It is therefore subsequently planned to study proteins which can integrate further into the network in phase with the development of drugs from biotechnology.

The properties of absorption, desorption and modeling will be continued as part of the thesis project*: https://www.theses.fr/s353389
*Camille Mathieu is the recipient of the funding award for her three-year-PhD thesis at 50% by the ED432 Doctoral School https://edsmi.hesam.eu/ and 50% by the EBI Social Fund https:// www.helloasso.com/associations/ecole-de-biologie-industrielle/collectes/lancement-de-la-fondation-ebi

 

Pictogramme La Vague 80 A3P

Camille MATHIEU – LABORATOIRE PIMM et EBI

 

 

Pictogramme La Vague 80 A3P

Emmanuel RICHAUD – LABORATOIRE PIMM

 

 

Pictogramme La Vague 80 A3P

Samar ISSA – EBI

Profil on

 

 

Références

✻
  • 1. Pharmaceutical Drug Delivery Market by Route of Administration (Oral, Injectors, Implantable, Syrups, Gels, Pulmonary, Solutions, Tablets, Syringes), Application (Cancer, Diabetes), Facility of Use (Hospitals), COVID-19 Impact – Forecast to 2026; MarketsandMarkets, 2021.
  • 2. Vargason, A. M., Anselmo, A. C., & Mitragotri, S. (2021). The evolution of commercial drug delivery technologies. Nature biomedical engineering, 5(9), 951-967.
  • 3. Sung, Y. K., & Kim, S. W. (2020). Recent advances in polymeric drug delivery systems. Biomaterials Research, 24(1), 1-12.
  • 4. Zhou, W., Lei, S., Liu, M., Li, D., Huang, Y., Hu, X., Yang, J., Li, J., Fu, M., Zhang, M., W ang, F., Li, J. Men, K. & Wang, W. (2022). Injectable and photocurable CAR-T cell formulation enhances the anti-tumor activity to melanoma in mice. Biomaterials, 291, 121872.
  • 5. Heskins, M., & Guillet, J. E. (1968). Solution Properties of Poly(N-isopropylacrylamide). Journal of Macromolecular Science: Part A – Chemistry, 2(8), 1441-1455.
  • 6. Tamai, Y., Tanaka, H., & Nakanishi, K. (1996). Molecular dynamics study of polymer− water interaction in hydrogels. 2. Hydrogen-bond dynamics. Macromolecules, 29(21), 6761-6769.
  • 7. Furyk, S., Zhang, Y., Ortiz-Acosta, D., Cremer, P. S., & Bergbreiter, D. E. (2006). Effects of end group polarity and molecular weight on the lower critical solution temperature of poly(N-isopropylacrylamide). Journal of Polymer Science Part A: Polymer Chemistry, 44(4), 1492–1501.
  • 8. Halperin, A., Kröger, M., & Winnik, F. M. (2015). Poly( N ‐isopropylacrylamide) Phase Diagrams: Fifty Years of Research. Angewandte Chemie International Edition, 54(51), 15342–15367.
  • 9. Lapointe, J. (2012). Fabrication et caractérisation d’hydrogels thermosensibles pour des applications de livraison ciblée de médicament et d’embolisation. Ecole polytechnique de Montréal.
  • 10. International Conference on Harmonisation (ICH) of technical requirements for registration of pharmaceuticals for human use (Pharmaceutical development Q8 (R2) https://database.ich.org/sites/default/files/Q8_R2_Guideline.pdf

Glossary & Abbreviations

✻
  • API – Active Pharmaceutical Ingredient
  • APS – Ammonium Persulfate
  • ATR – Atténuated Total Reflectance
  • BIS – N, N’ – méthylènebisacrylamide
  • CAR-T cells – Chimeric Antigen receptor (CAR) inserted into the T cells
  • DDS – Drug Delivery Systems
  • EDI – Deionized Water
  • GPC – Gel Permeation Chromatography
  • ICH – International Council for Harmonization
  • IRTF – Fourier Transform InfraRed
  • LCST – Lower Critical Solution Temperature
  • MTZ – Métronidazole
  • NIPAm – N-isopropylacrylamide
  • PLA – PolyLactic Acid
  • PLGA – Poly-Lactic-co-Glycolic Acid
  • PMMA – Poly (MéthylMéthAcrylate)
  • PNIPAm – Poly(N-isopropylacrylamide)
  • Tc – Critical phase transition temperature
  • TEMED – N, N, N’, N’-TétraMéthylEthylèneDiamine
  • THF – TétraHydroFurane
  • UV-Vis – Ultraviolet-Visible Spectroscopy

 

Share