Differential Analysis of O-(2- hydroxypropyl) cellulose by Using Two-Dimensional 1H-NMR Spectroscopy

Two-dimensional 1H-NMR is used to determine the intra-molecular interactions of O-(2-hydroxypropyl) cellulose (HPC) in aqueous (D2O), DMF and DMSO solutions. Four grades HPC with different molecular weights are analyzed by using NOESY (Nuclear Overhauser Effect Spectroscopy) for proton-proton cross-interactions. A strong dependence of the polymer chain structure on the HPC Molecular Weight (MW) is overserved. The lower MW HPCs exist in solutions as a more linear chain showing less proton-proton interactions whereas the higher MW HPCs are more twisted and bended and form a tangled molecule mess with very intensive interactions between the -CH3, -CH2and -C-H protons. From all the grades, the ultra-low molecular weight HPC-UL (MW 20,000) revealed the weakest proton-proton cross-relaxations and exists in solutions probably only as an almost linear chain polymer. Research Article Differential Analysis of O-(2hydroxypropyl) cellulose by Using Two-Dimensional 1H-NMR Spectroscopy Naotaka Sakamoto1, Edmont Stoyanov2* 1Nippon Soda Nihongi, 950 Fujisawa, Nakago-ku, Joetsu, Niigata 949-2392, Japan 2Nisso Chemical Europe, Berliner Allee 42, 40212 Duesseldorf, Germany Received: 19 December, 2019 Accepted: 28 February, 2020 Published: 02 March, 2020 *Corresponding author: Edmont Stoyanov, Nisso Chemical Europe, Berliner Allee 42, 40212 Duesseldorf, Germany, Tel: +49-211-130 69 473; Mobile: +49-151-20 03 01 88; Fax: +49-211-32 82 31; E-mail: ORCiD: https://orcid.org/0000-0002-1571-9259


Introduction
The solubility enhancement of poorly soluble drugs is known to be one of the biggest challenges in the pharmaceutical technology. The permanently increasing number of APIs (Active Pharmaceutical Ingredient) with low solubility creates the need of new polymers that enhance the API solubility and bioavailability. Usually used as a binder, the O-(2-hydroxypropyl) cellulose (Hydroxypropyl Cellulose, HPC) revealed a new and unexpected activity as solubility enhancer for poorly soluble drugs. Recent papers described the successful application of HPC as solubilizing polymer for nano-suspensions and amorphous solid dispersions [1][2][3]. Ito and Konnerth [1,2], described in two papers the application of HPC and copovidone for the preparation of nano-suspensions where the best results were achieved with the low molecular weight HPC-SSL. Zecevic et al., [3], reported the application of HPC-SSL in combination with HPMCAS as a solubilizer for Dipyridamole and Griseofulvin, two poorly soluble drugs with rash precipitation under certain pH conditions. Prodduturi et al., [4], reported an investigation of different molecular weight HPCs in Hot Melt Extrusion (HME) with clotrimazole where the lowest MW HPC demonstrated better dissolution results than the higher MW HPCs. This was explained with the possible increased entanglement of the polymer chain and a slower polymer erosion. Ten years later Osawa et al., [5] and Sarode et al., [6], gave the same explanation for the better solubility and permeability results of sulindac [5] and felodipine [6], in HPC-SSL containing solid dispersions. A short time ago, some of us published the effi cient performance of the ultra-low molecular weight HPC-UL as best milling aid and solubilizer for Izraconazole in aqueous nano-suspensions [7].
The reason for the much better functionality of the low mwt HPC as solubilizer for poorly soluble drugs is still not well clarifi ed and a close look at the HPC structure in solution could give the answer of this question.
The chemical structure of hydroxypropyl cellulose was already investigated per 1 H and 13 C-NMR [8,9]. Kimura et al., [8], described detailed the Degree of Substitution (DS), Molecular Substitution (MS) and even the different reactivity of the hydroxyl groups in the cellulose molecule based on the 13 C-NMR in D 2 O peaks integrals. By using the same carbon NMR method, Desai et al., [9], explained the different performance of HPCs with different cloud points also giving details on the ratio outer/single carbons vs inner carbons. These fundamental works assume the HPC molecular structure as independent of molecular weight and that the grade examined in the studies was assumed to be representative of the entire family of HPC molecular weights.
Based on the published information we cannot explain why only the low molecular weight HPC works as a drug solubilizer. No meaningful differences in the physico-chemical properties (solubility, DS, MS and inner/outer carbon ratio) between the different grades of this cellulosic ether can be found. In order to get better understanding in this phenomenon, we decided to investigate what happens with the dissolved HPC polymer chain in the space by using a special two-dimensional 1 H-NMR analysis -Nuclear Oherhauser Effect (NOE). Standard 1 H-NMR spectra show the proton spin-spin coupling due to chemical bonds, whereas NOE occurs through the space when the distance between two protons is less than 4.9 Å. Thus, by using NOESY (Nuclear Overhauser Effect Spectroscopy) we can gain information on atoms and groups that are in close proximity to each other in the same molecule. This NMR method has been already successfully used for the investigation of three-dimensional structures of many proteins and other macromolecules [10][11][12][13][14].
In the present work, we describe a differential analysis of four grades hydroxypropyl cellulose with different molecular weights (viscosities) by using NOESY in DMSO-d 6 , DMF-d 7 and D 2 O.

Chemicals
Hydroxypropyl cellulose (HPC) with average molecular weight of 140,000 (HPC-L), 100,000 (HPC-SL), 40,000 (HPC-SSL) and 20,000 (HPC-UL) were provided by Nippon Soda, Japan. All the HPC grades have similar physicochemical characteristics like Molecular Substitution (M.S.), Hydroxypropoxy Content (HPO) and outer methyl+single methyl to inner methyl ratio [6] (outer/inner) were selected for the measurements ( Table 1). The only relevant difference is in the Molecular Weight (MW).

Sample preparation
For DMSO-d 6 and DMF-d 7 , in order to avoid the OH groups exchange with residual H 2 O, the samples were prepared as follow: a. preliminary weighted amount of HPC was dried at 45°C overnight, then placed in a desiccator over CaCl 2 for 30min under vacuum; b. the sample was then dissolved in the deuterated solvent (0.5%) at room temperature under nitrogen atmosphere. For deuterated water (D 2 O), where the residual H 2 O doesn't play an important role, the sample preparation involves weighting and preparing of a 0.5% solution.

Equipment & measurement conditions
Bruker Biospin AVANCE III HD 500 Onebay was used for the measurements: mixing time 0.3s; number of scans 8.
Tetramethyl silane (Me 4 Si) was used as an internal standard.

Results and discussion
The hydroxypropyl cellulose is a cellulosic ether that is composed of approximately 25-30% cellulose and 70-75% polypropylene oxide (HPO content Table 1, Figure 1).   Table 2.  Figure 5 shows the 1 H-NMR of HPC-L as example.
All other HPC grades (SL, SSL, and UL) have identical 1 H-NMRs with HPC-L.

NOESYs of HPC-L, SL, SSL and UL NOESYs in DMSO-d 6
The HPC with the highest molecular weight (L) showed multiple H-H interactions between the functional groups ( Figure 6). The strongest was the one between the protons of CH, CH 2 and CH 3 groups (III and IV). This interaction was very strong, whereas it is independent on which proton was is the source. This speaks for a close proximity pf those groups in the space. The methyl protons demonstrated also interaction with the C1 protons form the cellulose moiety (II) as well as with the protons of the hydroxyl groups (I). This speaks for close distance between the inner and outer propoxyl groups and cellulose skeleton.    The lower molecular weight grades HPC, revealed the same interactions however with decreased intensity, proportionally to the decrease of their molecular weight (Figure 7).
Generally, the intensity of all observed cross-relaxations (I-IV) was decreased with the decrease of the HPC molecular weight. The biggest difference was observed in the crossrelaxations between the hydroxyl and C1 cellulose protons and methyl group protons. We can conclude that the distance between the end-propoxy groups and cellulose skeleton is bigger at the lower molecular weight HPC. At HPC-UL (MW 10,000), the cross-relaxation was with the lowest intensity speaking for an almost linear, not much bended structure in DMSO solution.

NOESYs in DMF-d 7
The 1 H-NMRs in DMF-d 7 were almost identical with those in DMSO-d 6 . Differences were observed in the NOE spectra where the proton-proton cross-relaxations in DMF were much stronger ( Figure 8).
In DMF-solutions, the HPC molecule seems to be more tangled than in DMSO-solutions. Thus, in DMF, the functional groups appear to be in closer proximity in the space and interact stronger compared to DMSO. This is also confi rmed by the NOESYs of the other HPC grades (SL, SSL and UL). As shown in      In all solvents used, the ultra-low molecular weight HPC-UL revealed the lowest intensity of the proton-proton crossrelaxation confi rming a less tangled and bended structure in solutions compared with the other, higher MW HPC grades (L, SL and SSL, Figure 12).
In aqueous solutions, the observed differences in the intensity of the CH 3 -CH 3 interactions (proton-proton crossrelaxations) at 1.1ppm caused by closer proximity of this functional groups in aqueous solution, confi rm the observed minor differences in the cloud point of the different HPC grades. A stronger CH 3 -CH 3 interaction with increase of the HPC molecular weight corresponds to a decrease of the cloud point ( Figure 13).