Physicochemical Characterization of Nanobiocomposites

Abstract

Nanobiocomposites (NBCs) have many applications in drug delivery, tissue engineering, etc. The need for NBC physicochemical characterization is mandatory before investigating their usefulness in developing drug delivery systems. This chapter will explore the basic and the most recent techniques used in the physicochemical characterization of these biocomposites. Examples of physical properties include morphological properties using microscopy (size, porosity, etc.), particle size analysis and surface charge, powder X-ray diffraction, thermal, mechanical, and rheological properties, etc. Examples of chemical properties include molecular weight determination, solubility and purity assessment, degree of functionalization, and gelling properties, using spectroscopic techniques (UV, MS, NMR, etc.). For each property, the following points will be elucidated: sample preparation, factors affecting the accuracy of the test results, examples of data interpretation from the recently published literature, and test limitations, if any.

1. Introduction

Biodegradable polymers offer great potential in drug delivery using nano-scale systems. However, natural polymers are more attractive for pharmaceutical applications as they have sustainable resources, low toxicity, biocompatibility, biodegradability, and the ability to be modified, allowing tuning of their properties to suit their application in the pharmaceutical field [1]. Many polysaccharide polymers have been investigated for drug delivery application, including chitin/chitosan, agarose, Bacterial cellulose, gum arabic, tragacanth, alginate, gellan gum, starch, carrageenan, dextran, nanocellulose, and Xanthan gum [2].

On the other hand, biocomposite materials consist of at least two components, including a continuous matrix phase, which is usually a natural polymer, and discontinuous reinforcement material, which will be used to reinforce the backbone of the biopolymer. Polymers with counter ionic properties like cellulose and chitosan or using a crosslinker like tripolyphosphate (TPP) are commonly used [3]. Reinforcement can be performed using physical crosslinking methods, which typically include electrostatic/ionic interactions, hydrophobic interactions, and π–π stacking interactions. In comparison, chemical crosslinking methods are typically covalent crosslinking, which h can be a direct crosslinking or free-radical polymerization [4, 5]. For example, the fabrication of chitosan NPs using tripolyphosphate crosslinking, followed by covalent crosslinking using coupling chemistry [6, 7]. As a result, the size and properties of the NBCs offer excellent features in the drug delivery field [8]. In addition, micro to nano particles, can be prepared by a variety of technologies, including the Innotech Encapsulator, ionic-gelation techniques, vibrational jet-flow technology, dripping, and interphase technique approach [9], can be used to create the tiny micro- and nanocapsules [10, 11]. On the other hands, nanogels can be prepared in uniform gel sheets or in macro disks using the parallel plates equipment [12].

In nanocomposites, the interaction between matrix and reinforcement is very high due to the high surface-to-volume ratio [8, 13]. The improved properties of nanocomposites depend on the properties of each component, their relative amounts, and the overall geometry of the nanocomposites. Generally, when natural polymers are utilized in the fabrication of NBCs, enhanced properties, such as NP size and surface charge, mucoadhesiveness, adsorptive, etc., can be achieved [1, 3]. These characteristics can be optimized to suit a wide range of applications in non-conventional routes of administration, including nasal, rectal, buccal, etc. In addition, many of these NBCs have been fabricated as stimuli-responsive drug delivery systems (pH, temperature, light, etc.) [14, 15].

This chapter focuses on the physicochemical characterization of polysaccharides-based biocomposites investigated as nano drug delivery systems. Before utilizing a new polymer in NBCs fabrication, a complete characterization should be performed using reliable and validated methods. Then, biocomposites can be characterized using traditional and advanced methods, including spectroscopic techniques like Infrared (IR), Nuclear Magnetic Resonance (NMR), etc. Additional properties like thermal, mechanical, rheological, gelling, adsorptive, etc. are also part of the characterization procedure commonly reported. Representative examples from the most recently published literature will also be discussed. Figure 1 proposes a plan to follow in the physicochemical characterization of natural polymers and their corresponding NBCs.

Figure 1.

A proposed approach for the physicochemical characterization of natural polymers and their respective nanobiocomposites (NBC).

2. Chemical characterization

2.1 Ultraviolet: Visible (UV: VIS) spectroscopy

UV–VIS analysis is a simple, low-cost, and rapid technique that is based on exposing the biocomposite sample to electromagnetic radiation in the UV–Vis region (typically 190 to 900 nm). The UV range usually extends from 100 to 400 nm, and the visible range is approximately 400 to 800 nm. Upon exposure to light, molecules will absorb or transmit light, depending on the chemical composition of the irradiated material. A specific spectrum is generated with a specific wavelength corresponding to the characteristic functional group of the scanned biocomposite [16]. Factors affecting spectral characteristics are the chemical composition and sample light scattering properties related to its microstructure. In order to use this method, the sample should have functional groups that can absorb light in the UV–VIS region, for example, an aromatic ring. This method also offers the rapid monitoring of changes in the biocomposite when exposed to variable pH and temperatures [17].

Cazón et al. employed the UV technique to optimize the composition of films based on vegetable and bacterial cellulose combined with chitosan and polyvinyl alcohol (Figure 2). In addition, with the application of mathematical and statistical, they were able to quantitatively estimate the composition of each component with high accuracy based on the data generated from UV–VIS–NIR spectra. Interestingly, the proposed method enabled the discrimination of the geographic origin of the investigated biopolymers [16].

Figure 2.

UV–VIS region spectra of the bacterial cellulose samples with chitosan and polyvinyl alcohol. MCQP is variable composition of bacterial cellulose with chitosan and polyvinyl alcohol [16].

Niroomand et al. used UV–visible spectroscopy to measuring the optical transmittance and opacity of the pure cellulose and Nano-chitosan/cellulose films, by scanning at 200–800-nm wavelengths using Eq. (1) [18]:

$$\mathit{\text{Opacity}}=1/4\frac{\mathit{\text{Absorbance at}}600\mathit{nm}}{\mathit{\text{Film thickness}}\left(\mathit{mm}\right)}$$E1

A high transmittance indicates film transparency. Additionally, the researchers observed a slight increase in the film opacity at high dosing rates reaching 15% of nano-chitosan particles, which can result from partial agglomeration of NBCs [18].

2.2 Fourier transform-infra red (FT-IR) and attenuated total reflection (ATR)

In order to use Infrared spectroscopy (IR) as a characterization tool, NBC molecules must absorb light in the infrared region of the electromagnetic spectrum, converting it to molecular vibration. This absorption is measured as a function of wavelength (as wave numbers, typically from 4000 to 600 cm⁻¹). This absorption is characteristic of a sample’s chemical bonds (stretching, binding, etc.) [19]. Using a mathematical algorithm, the wave number raw data is transformed into an IR spectrum that serves as a characteristic “molecular fingerprint” that can be used in the structural identification of organic samples. A solid sample is either ground with IR potassium bromide (KBr) and pressed into a transparent disc or is thinly sliced and placed onto a KBr window. While liquid samples are directly measured or diluted with an IR transparent solvent [19, 20].

Other IR techniques based on reflection rather than transmittance are Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS)and Attenuated Total Reflection (ATR). In DRIFTS, the IR light only interacts with the surface of a material to collect chemical information following mixing with KBr. Besides being a non-destructive method, ATR is a simple handling technique that utilizes a limited amount of the tested sample that is directly placed in a zinc selenide (ZnSe) crystal or diamond without any other ingredient. It can be used to study soft, stiff, and rigid polymers. However, ATR has the drawback of generating false data. ATR interrogates the surface of the sample so that the chemistries on the surface are amplified. The refractive index determines the strength of the reflection, so wherever an absorption band is present, the extent of the reflection will change [21]. Generally, it is essential to examine the unmodified polymer and each NBC component separately before scanning the NBC itself. This allows the observation of new bands, changes in band intensity, etc., that can be characteristic of the NBC. In addition, caution should be made to avoid non-uniform particles or large particles that can affect the data generated in the DRIFTS and ATR. Some disadvantages include the interference of water, CO₂ effect, etc. [22].

IR has been widely applied in measuring grafted polymers’ functionalization prior to NBC fabrication [23]. Figure 3 shows the IR spectrum of acrylic functionalization of cellulose nanocrystals with 2-Isocyanatoethyl Methacrylate(IEM). Strong absorptions at 1723 and 1640 cm⁻¹ indicated the attachment of C〓O and C〓C groups on modified cellulose nanocrystals (mCNCs). The disappearance of the −NCO peaks from 2-isocyanatoethyl methacrylate and the appearance of the multiple absorption peaks between 1200 and 1700 cm⁻¹ is associated with the formed urethane linkage between cellulose nanocrystals and IEM. A small increment of the absorption between 2900 and 3000 cm⁻¹, and the increased C▬H stretch of the methyl group of IEM indicates functionalization [24]. Similar studies that reported the use of IR in confirming functionalization include alginate [25], methoxylated pectin and amidated LMP [13], chitosan functionalization with the PNIPAAm [26], cellulose-propargylated chitosan [27], Melamine-Functionalized Chitosan: [28], etc.

Figure 3.

ATR-FTIR spectra of Unmodified Cellulose nanocrystals (umCNC), modified Cellulose nanocrystals (mCNC), and 2-isocyanatoethyl methacrylate (IEM) [24].

IR can be used to study crosslinking within NBCs. For example, Stanescua et al. reported using FT-IR to study crosslinking in NBC based on chitosan/bacterial cellulose used as a wound dressing. FTIR spectra show three new peaks in the regions 2980, 2972, and 1425 cm⁻¹, which can be attributed to the asymmetric stretching vibration of C▬H from methyl groups and for bending vibration of C▬H from methyl groups of the grafted chitosan, respectively (Figure 4). These vibrations suggest the generation of a new physically crosslinked network via hydrogen bonding [29].

Figure 4.

FTIR spectra of uncrosslinked and physically crosslinked chitosan (individual component) [29].

In addition, IR can be used to confirm the immobilization of biomolecules onto NBCs by observing the new bands or changes in the IR band pattern representing specific chemical groups. İlgü et al. investigated the immobilization of recombinant esterase onto chitosan nanoparticles (NPs) by physical adsorption under several immobilization conditions. As seen in Figure 5, the chitosan nanoparticles (NP) and the enzyme immobilized chitosan NP spectra ((C) and (D)), where the peak intensity at 1650 cm⁻¹ has increased, while two peaks shifted from 1560 to 1550 cm⁻¹ and from 1413 to 1407 cm⁻¹. The strength of these two peaks’ intensity also decreased dramatically. These changes in the FTIR spectrum confirmed the immobilization of esterase on chitosan NPs [30].

Figure 5.

FT-IR spectra of (A) chitin, (B) chitosan, (C) chitosan NP, (D) enzyme immobilized chitosan nanoparticles [30].

2.3 Nuclear magnetic resonance (NMR)

This technique is widely used in identifying and characterizing novel functionalized polymers and their respective NBCs. It gives some idea about the NBC chemical structure, and morphology, for example, the amount and orientation of crystalline phases in semi-crystalline NBCs and the domain sizes in phase-separated polymeric NBCs [31].

NMR is based on exposing a charged nucleus like hydrogen (¹H NMR) or carbon (¹³C NMR), etc. to a strong magnetic field, which allows the transfer from a low energy state to a high energy state corresponding to a certain radio frequency. The energy is then emitted at the same frequency when the spin returns to its base level. Capturing these signals will give an NMR spectrum with characteristic chemical shifts for the spinning nucleus [32]. The precise resonant frequency of the energy transition is affected by electron shielding, which in turn is dependent on the chemical environment (i.e., the functional group within a polymer). The presence of an electronegative group around the nucleus will result in a higher resonant frequency in general. In order to get accurate measures using this technique, the following factors should be considered: signal-to-noise ratio, saturation effects, peak shape, resolution, isotopic satellite, spinning sidebands, baseline slant, and curvature [33, 34]. The advancement of NMR to include computer-assisted methods enabled more information on molecular modeling and conformational analysis of many natural polymers [33].

NMR can be used to characterize liquid NBCs samples like gels, dispersion, melt, and solutions with increased spectral specificity compared to solid samples [31, 32]. Consequently, dilution, dispersion, increased temperature, etc., can give more information related to polymeric microstructure, its dynamics, and interactions with other ingredients within an NBC [33]. Liquid state NMR can be classified into one-denominational and multi-dimensional (2D and 3D) techniques. Hyphenated techniques include On-line HPLC-NMR, Supercritical Fluid Chromatography (SFC)-NMR, and Offline capillary electrophoresis. Isotopic labeling (¹³C, ^l9F, ¹⁵N, and ³¹P) can facilitate relaxation studies of polymers hence, enabling studying crystallization within these polymers. Nano-NMR can be used to study heterogeneous and limited quantity samples [31, 35].

On the other hand, the solid-state NMR spectrum tends to have broad lines because of chemical shift anisotropy and dipolar and quadrupolar couplings. High-power dipolar decoupling, cross-polarization, and magic angle spinning to produce high-resolution ¹³C NMR spectra avoid long instrument running time. Likewise, the combined rotation and multi-pulse (CRAMPS) experiment can permit H spectra with narrower line widths to be obtained [31, 33, 35].

NMR has been extensively used to determine the degree of functionalization of many natural polymers and to characterize their corresponding NBCs through careful measurement of peak heights or areas under the signal peak in the NMR spectrum using a suitable reference standard. Examples of published literature about using ¹H NMR include: include N-carboxyethyl chitosan and glycol chitosan [36], 2,3-epoxypropyltrimethylammonium chloride grafted starch [37], heteroaryl pyrazole chitosan derivatives [38], sulfonated chitosans [5], Cellulose Nanocrystals with 2-Isocyanatoethyl Methacrylate [24] and many others. 1H NMR has also been used to predict the rigidity of polymers and different phases of a polymer like cellulose [33]. The following paragraphs will discuss some of these published data.

Zhou et al., 2022 et al. used NMR spectroscopy to study the functionalization of chitosan and to detect the suitable pH that enables optimum functionalization (Figure 6). The location of the chemical shift of the aliphatic portions of the chitosan and the aromatic protons of the grafted substituent is a confirmation of the grafting procedure. Interestingly, signal intensity at δ 6.823 ppm (typical aromatic proton signal of substituent) was dependent on the pH of the medium, i.e., higher substitution was observed at pH 6.4 compared to pH 3.4 [39].

Figure 6.

1H NMR spectra of sinapic acid (SA), chitosan (CS) and sinapic acid-graft-chitosan (SA-g-CS) conjugates synthesize under different pH conditions (3.4–6.4) [39].

NMR can also be used to monitor the start and end of the gelation process in NBCs and to predict stability over time. Craciun et al. reported the preparations of a high water content chitosan-based hydrogel that was monitored in deuterated water over 22 days at room temperature (Figure 7) Gelation was driven by the formation of an imine group between chitosan (NH₂ group) and vitamin B6 precursor, pyridoxal 5-phosphate (aldehyde group). The beginning of the gelation process was evidenced by the appearance of the chemical shifts of the imine group, while the progressive diminishing of the integrals of imine and aldehyde protons and the appearance of the enol proton (around 6.5 ppm) indicated the end of the process. This suggests that a too diluted system favored the shifting of imination to the reagents and the stabilization of the enol form of aldehyde. Consequently, the water content of this hydrogel can limit the storage duration at room temperature to less than 22 days [40].

Figure 7.

Representative 1H-NMR spectra of hydrogels (a) with higher water content 1q, 2 t and (b) lower water content 3d, 4d, recorded over time (from up to down: 1, 7, 15, and 22 days) [40].

Heinze et al. used ¹³C NMR to identify the functional groups following grafting 2,3-epoxypropyltrimethylammonium chloride to starch. The ¹³C NMR in Figure 8A shows different carbons type in the backbone of the starch and its grafted conjugate. Additionally, the researchers used Distortionless Enhancement by Polarization Transfer (DEPT) NMR to confirm the connections between the carbons. DEPT can be easily combined with a 1H isotropic-chemical-shift filter that selects NCH/OCH signals versus CCH(C, C) signals (Figure 8B) [37].

Figure 8.

(A) The 13C-NMR spectrum of cationic starch sample H 6 (degree of substitution (DS) =0.66) was measured in D2O at 607C (R = H or cationic group according to DS). (B) DEPT-135 spectrum of cationic starch P 3 (degree of substitution = 1.05) [37].

On the other hand, 2D NMR like COSY (COrrelated SpectroscopY, H-H NMR) and HSQC (cross-polarization heteronuclear single-quantum coherence, H-C NMR) can be used to study correlations between two nuclei which are separated by one bond like two hydrogens or hydrogen and carbon within a chemical structure. Since all NBCs have carbon and hydrogen atoms, either in the grafted molecules or in the host structure itself, 2D-NMR can be a valuable tool for studying the structural interactions in NBCs [32]. The COSY technique involves plotting ¹H NMR for each component to detect the proton-proton interaction, which is then plotted as 2D contours in the XY plane to detect interaction dynamics. Wang et al. used 2D-NMR (COSY) to study drug-polymer interactions of N-succinyl chitosan-alginate grafted NPs loaded with mangiferin (an anti-atherosclerotic drug). As seen in Figure 9A, the drug and N-succinyl chitosan interact at several neighboring protons; for example, the phenolic –OH of mangiferin (δ 9.25) interacts with the –COOH proton of the succinyl side chain (δ 12.38).and the –OH of mangiferin and –C▬O, –NH–, carboxyl of side chain –NHC– – OCH₂CH₂COOH in N-succinyl chitosan [41]. On the other hand, HSQC(¹H▬¹³C) can also be used to study the interaction between hydrogen and carbon atoms within a biopolymer. Huamani-Palomino et al. utilized the HSQC spectrum to confirm the purification process of alginate (Figure 9B) by observing the coupling bands of the alginate monomers (glucoronic acid and mannuronic acid) [25].

Figure 9.

(A)2D NMR (COSY) spectra of mangiferin loaded NSC-alginate formulation. C-denotes proton peaks for N-succinyl chitosan and m-depicts proton peaks for mangiferin [41]. (B) HSQC spectrum of purified alginate (AlgP) [25].

2.4 Powder X-ray diffraction (XRD/PXRD)

It is well established that natural polymers show a variable proportion of their disordered amorphous regions and ordered crystalline regions, which consequently affect the characteristics and applications of their NBCs. The presence of amorphous regions highly affects the polymer plasticity and flexibility, while the crystalline regions affect the elasticity and stiffness of these materials [42]. The amorphous/crystalline proportion of a natural polymer and their respective NBC are greatly affected by purification and drying (solvent evaporation, lyophilization, etc.) [43]. X-Ray diffraction (XRD) is an analytical technique that is used to determine the solid state, crystal size and shape, and phase identification with quantitative phase analysis of materials. The theoretical basis of X-Ray diffraction stands on Bragg’s Equation (Eq. (2)) [43]:

$$\mathrm{n\lambda }=\text{2d sinθ}$$E2

Where n is the order of reflection n = (1, 2, 3,….) λ, the wavelength, d the distance between parallel lattice planes, and Ө the angle between the incident beam and a lattice plane, known as Bragg angle [44]. The geometry of the crystal lattice determines the position of the peaks in an X-ray diffraction pattern. In general, as the material became more symmetrical, the peaks became fewer in its diffraction pattern. The peak intensities associated with the diffraction intensity are determined by the arrangement of atoms within the crystal lattice [45].

Experimentally, there are two methods of XRD, the Laue method, where θ is kept constant and λ varied, and the powder diffraction method, where λ remains constant, and θ is varied. In both methods, the intensity of the diffracted X-ray beam against diffraction angle 2θ is measured, which gives the diffraction pattern of the material. The pattern obtained in crystalline materials shows sharp maxima, called peaks, at their respective diffraction angle, and in amorphous solids, the orderly structure is absent, which gives rise to broad maxima called a hump [42]. X-ray scattering provides structural information at three different length scales by performing scattering experiments at such as 1 (wide XRD), 10 (small XRD), and 100 nm (ultrasmall XRD) angles. Natural polymers in general are not fully crystalline; so XRD is used to measure their degree of crystallinity [45]. Three important information are needed when interpreting an XRD diffractogram:

1. The position of the diffraction peaks.

2. The peak intensities.

3. The intensity distribution as a function of diffraction angle.

Prior to performing any XRD measure for an NBC, it is essential to scan each component alone (drug alone, polymer alone, crosslinker alone, etc.) followed by a scan of the physical mixture of two or more components, and finally, the NBC in order to compare the molecular interaction (Figure 10) [6]. Any peak position or intensity change indicates an interaction between the drug and the polymer upon NBC fabrication. At the same time, the broadening of peaks (halo-pattern) or decreased intensity of a peak indicates amorphous transition or the presence of an amorphous state [45].

Figure 10.

XRD spectrum for chitosan polymer (CS), crosslinker (STPP), Mebeverine Hydrochloride(MB.HCl), and Chitosan nanoparticles loaded with MB.HCl (CS + MB. HCl + STPP) [6].

Drug-polymer interaction within NBCs can also be studied by detecting the presence or absence of new peaks. For example, the XRD of mebeverine(MB) loaded chitosan NPs shows broad peaks indicating an amorphous state within the polymer or the NP (Figure 10). Moreover, the absence of additional peaks indicates the purity of the formulations. Bragg Law was used to calculate the crystallization of the chitosan polymer crystal practices, and it reached 4.5. Upon comparing the XRD of the NPs and that of each component, it can be concluded that the peak at 2ϴ = 26.9o is due to the sodium tripolyphosphate (STPP) crosslinker-drug interaction, while the peak at 2ϴ = 17.9o is related to the loaded drug mebeverine. Additionally, the absence of any additional peak indicates no change in the degree of crystallinity of the polymer during the fabrication of the NPs [6].

Similarly, Shahid et al. used XRD to predict the type of interaction within Ticagrelor-loaded chitosan-based NPs [46]. Ahmad et al. reported the use of XRD to study the crystallite structure within starch-based NPs prepared using mild alkali hydrolysis and an ultra-sonication process. Using the quantitative measurement of the area under the amorphous region and diffraction peaks the researchers concluded a decrease in crystallinity. The increased amorphous region was accompanied by diminished diffraction peaks following the size reduction of starch to the nanoscale [44].

The stability of NBCs during storage can also be assessed using XRD through the evaluation of their crystallinity over time. Burapapadh et al. evaluated the degree of crystallinity of itraconazole (ITZ) pectin-loaded NPs. Figure 11A shows that pectin alone exhibited a halo pattern indicating the amorphous state of the polymer, while the sharp peaks (17.45 and 17.95 (doublet), 20.30, and 23.45 2θ) of the drug alone suggest its high crystalline nature. The XRD patterns of drug-polymer physical mixtures showed similar peaks as untreated drug, indicating no change in drug crystallinity during the mixing process, while the XRD patterns of NPs showed the absence of the characteristic crystalline drug peaks (a typical broad hump of amorphous material), indicating that the drug is present on the noncrystalline form within the NPs. Likewise, the assessment of the XRD of the prepared sample after one-year storage at 25°C, showed (Figure 11B) the halo-pattern of the molecularly dispersed amorphous drug. However, there were some crystallinity peaks presented at approximately 12 and 21 2θ degrees. This indicates the start of transformation from amorphous to crystalline solid upon storage of the NPs for 12 months [43].

Figure 11.

(A) Powder X-ray diffraction patterns of ITZ, physical mixture of ITZ, and various types of pectin and nanoparticles prepared from nanoemulsion templates. (B) Powder X-ray diffraction patterns of various nanoparticles prepared from nanoemulsion templates, using a mechanical homogenizer, after 1-year storage at ambient condition (25°C) [43].

Small-angle X-ray scattering (SAXS) method utilizes smaller angles in scanning, typically from 0.1 to 10°, where the elastic scattering of X-rays caused by nanoscale structures in the polymer is recorded. This enables studying NBCs in the range of 0.5–100 nm up to 1000 nm. Such information includes size, shape, pore sizes, and other characteristic distances of partially ordered materials collected [36, 42]. Lin Y et al. reported sing SAXS coherent X-ray scattering (CXS) in studying the dynamics and gelation mechanism of glycol and carboxyethyl chitosan-based hydrogels with different dynamic interactions. In situ SAXS enables getting information about the nucleation and growth mechanism during the gelation process to form a hydrogel. Moreover, the continuous time-resolved CXS profile unveiled the dynamic behavior of different self-healing hydrogels in mesoscale, supported by rheological experiments [36].

2.5 Elemental analysis (EA)

This method is based on determining the molecular compositions by calculating the ratios of each element within a polymer, for example, carbon, nitrogen, hydrogen, etc., in addition to halogens. In the elemental analyzer, the sample is combusted at 1000°C in a special furnace. The analysis is accomplished by the quantities of CO₂, H₂O, and NO₂ produced by the combustion of the dried carbonaceous materials in excess oxygen. The weights of these combustion products are used to calculate the combustion of samples. The weight percentage of C and H is determined by infrared detection, whereas N content is measured by thermal conductivity detection [24, 47].

Li et al. reported using EA to detect the functionalization of glucose-conjugated chitosan nanoparticles (GCNPs). EA was used to determine the percentages of C, H, and N and the degree of N-succinyl glucosamine substitution (DS). The method is based on calculating the percentage ratio of the atomic mass of C, H, and N to the substituted and the unsubstituted chitosan [48].

One of the advantages of EA is the small amount of sample to be tested (5 mg). However, the sample should be as pure as possible and completely dry. Any impurities or trace solvents will interfere with the results and make the interpretation difficult. Ideally, solid samples should be tested in powder form. The analysis should be carried out under the nitrogen gas purge for air-sensitive samples.

Elemental analysis has been widely used to estimate the degree of functionalized pectin with Polyacrylamide [49], alkyl pectin [50], histidine-pectin [51]. Elemental analysis was also used to characterize cellulose grafted polymers: phenylacetic acid and hydrocinnamic acid [52], 2-propynoic acid, 4-pentenoic acid, 2-bromopropionic acid, or 3-mercaptopropionic acid [53], acryloyl cellulose [24]. Chitosan grafting with poly N isopropyl acrylamide [26], Melamine [28], 5-nitroisatin [54], mono- and di- sulfonic [5], maltol and ethyl maltol [38], cellulose beads [27], Alginic acid with cysteine [25], N-alkylamides, hydrazide, and hydroxamic acid [55]. Starch with 2,3-epoxypropyltrimethylammonium [37], 3- chloro-2-hydroxypropyl) trimethylammonium chloride [56], poly(methyl methacrylate-co-styrene [57]. Taubner et al. reported the elemental analysis of amidated alginic acid. Table 1 shows the content % of carbon, nitrogen, and hydrogen in addition to the degree of amidation of the various samples [55].

Sample	Reagent	Content (% m/m)			Substitution (mol%)
		N	C	H	Degree of amidation
1		0	32.85	5.20	—
2	methanol	0	38.01	5.39	—
3	n-butylamine	4.58	46.00	6.95	77.86
4	n-hexylamine	4.07	49.83	7.59	72.34
5	n-octylamine	4.08	52.98	8.20	83.96
6	n-dodecylamine	2.87	54.94	8.77	57.94
7	n-octadecylamine	2.62	58.11	9.12	76.55
8	ethylendiamine	7.79	34.02	6.24	73.24
9	ethanolamine	5.37	38.84	6.29	93.19
10	hydrazine	6.21	29.84	4.90	53.52
11	hydroxylamine	2.52	34.94	5.35	37.15

Table 1.

Organic elementary analysis of the alginic acid and its derivatives [55].

3. Physical characterization

3.1 Thermal analysis

These are a group of methods that examine changes in a solid sample when heated as a function of temperature and time. Information that can be obtained includes crystallinity (melting point), amorphous state (Glass Transition (Tg)), the heat of reaction (enthalpy (H)), thermal stability/degradation, etc. In this section, the most common thermal methods used in NBCs characterization will be discussed: Differential Scanning Calorimetry (DSC), Thermogravimetric (TGA), and Thermal Mechanical Analysis(TMA). It is well established that many features within a thermogram indicate certain transformations within NBCs as described in the following paragraphs [58].

3.1.1 Differential scanning calorimetry (DSC)

DSC determines the solid transitions as a function of temperature and time. It is used to identify solid–solid transitions (crystallization, polymorphism, etc.), melting, decomposition, and others [58]. In general, an initial transition that is observed in DSC is the solvent evaporation while the final thermal peaks can be due to polymer decomposition [58, 59]. Solid-state transitions can be detected following cooling of the sample by re-running the thermal analysis provided the sample is stable with no signs of degradation (change in color, gas evolving, etc.). Crystallization is a kinetic process that is detected by an exothermic peak in a DSC thermogram. The endothermic (heat absorption) and exothermic (heat released) peaks and magnitudes indicate the thermal phase transformation of the composites. The principal thermal data extracted from this analysis are the glass-transition temperature (Tg), degree of crystallization (Xc), crystallization temperature (Tc), and fusion temperature (Tm). The enthalpy variation and heat capacity of the composite can also be determined [60, 61].

3.1.2 Crystallinity and amorphous state of NBCs

Many natural polymers exhibit various degrees of crystallinity. DSC can differentiate these degrees by measuring the Glass Transition (Tg) which is the softening temperature characteristic of an amorphous state. This is attributed to the molecular mobility within the solid sample. This transition highly affects solubility, drug release, drug-polymer interaction, stability during storage, and many other physical properties.

Figure 12 shows DSC thermograms of physical mixtures of high methoxyl (HM) pectin or low methoxyl (LM) pectin and amidated LM pectin (ALMP) used to deliver itraconazole(a poorly water-soluble drug) and their respective NBCs. The thermal properties of physical mixtures of itraconazole and various types of pectin at a ratio of 1:6 were compared to those of their respective NPs. The melting peak of itraconazole crystals can be observed in the first three physical mixtures with an endothermic peak at 166–168°C. However, following encapsulation within the NP, this peak disappeared, indicating molecular dispersion of the drug within the polymer [43].

Figure 12.

DSC thermograms of physical mixture of itraconazole and high methoxyl (HM) pectin or low methoxyl (LM) pectin of pectin and nanoparticles [43].

Xiao et al. utilized DSC to evaluate a series of novel cellulose esters containing phosphorus, including cellulose diphenyl phosphate (C-Dp) and cellulose acetate (CA)–diphenyl phosphate mixed esters. Figure 13(A) depicts the Tg of the various grafted polymers compared to native cellulose, which does not exhibit a glass transition when subjected to heat. The thermogram shows a decrease in the Tg value with increasing the degree of substitution of cellulose which was attributed to disrupting the hydrogen bonding in the cellulose hydroxyl groups affecting molecular mobility within the cellulose chain [62].

Figure 13.

(A) DSC thermograms of unmodified cellulose, C-Dp (DS = 0.99) and C-A–Dp M1 - M5 with increasing degree of substitution [62]. (B) DSC of unmodified and acylated pectins with different acyl lengths (DS: 10–20%) [50].

Likewise, Zheng et al. evaluated the Alkyl pectin with various fatty acid (C4–C16) bromides using DSC. As shown in Figure 13B, with longer alkyl chain lengths (C8▬C16), the glass temperature peaks became higher while the peak areas became broader when compared to native pectin [50].

3.1.3 Thermogravimetric analysis (TGA)

TGA is used to study the change in the samples’ weight as a function of temperature. Weight loss includes water and solvent evaporation, decomposition, etc. TGA has been widely used to study the effect of varying the type and percentage of nanofillers within an NBC. Polymer decomposition, either in the presence of oxidative or non-oxidative gas, significantly depends on the presence of fillers and their dispersion scale [58]. On the other hand, Derivative thermogravimetry (DTG) is another useful technique that can be used to evaluate the thermal stability of NBCs. Hu et al. reported the use of TGA and DTG to study double-layer hydrogel based on sodium alginate (SA) -carboxymethyl cellulose (CMC) as a sustained drug delivery system. As seen in Figure 14A and B there are three major weight losses in hydrogels, each corresponding to a change in the nature of the sample. The first weight loss below 100°C is due to water desorption, the second one at 270°C is due to the destruction of glycosidic bonds within the hydrogel, and the third one at about 400°C is due to the destruction of outlayer polymer. Additionally, the incorporation of outlayer polymer(poly(N,N-dimethylacrylamide) (PDMA) or poly (acrylamide) (PAA)) into the hydrogel composition added to the thermal stability and some changes in the degradation pattern. Interestingly, the pronounced delay in the degradation phase (third phase) was attributed to the inclusion of the synthetic polymer (poly(N,N-dimethylacrylamide) (PDMA) or poly (acrylamide) (PAA) in the outer layer of the hydrogel formula [63].

Figure 14.

(A) The TG curves of on NBCs Based on sodium alginate (SA) -carboxymethyl cellulose (CMC) and (poly(N,N-dimethylacrylamide) (PDMA) or poly (acrylamide) (PAA), SA-CMC@PDMA-1, 2,3 and SA-CMC@PAA-1; (B) The DTG curves of SA-CMC@PDMA-1, 2,3 and SA-CMC@PAA-1 [63].

Chang et al. studied the stability of starch-based NPs upon using anionic, cationic, and amphoteric starch NPs. As can be seen in Figure 15A and B, starch-modified NPs exhibited a lower maximum degradation temperature compared to the maximum degradation temperature of unmodified starch NPs (310.83°C). The results indicated that the thermal stability of modified starch NPs (cationic, anionic, and amphoteric) decreased, evidenced by a lower decomposition temperature, compared to the non-modified starch NPs. This degradation was attributed to the intermolecular forces acting on the starch NPs [56].

Figure 15.

Thermogravimetry (A) and derivative thermogravimetry (B) of Unmodified starch NPs (SNPs), anionic (CMSNPs), cationic (CSNPs-1, CSNPs-2), Amphoteric (CM-SNPs-C-2, and CM-SNPs-C-1) [56].

Similar findings were reported by Kahdestani et al. during investigating the teicoplanin-loaded NPs based on chitosan/TPP. The TGA was used to evaluate drug and NP degradation over temperature changes. They reported a weight loss of 21.64% occurred between 127 and 226°C related to the removal of the residual water of the NPs and degradation of the polymer (Figure 16). Additionally, the authors attributed the increased stability of the NPs to the existence of phosphate groups (P〓O and P▬O) in chitosan NP leading to less degradation. However, TGA data showed a reduction in the thermal stability of chitosan NPs due to decreased crystallinity within the NPs compared to chitosan alone [64].

Figure 16.

TGA thermograms for (a) chitosan, (b) chitosan nanoparticles, (c) chitosan nanoparticles containing drug and (d) teicoplanin [64].

In another study, Kassab et al. reported the identification of the various decomposition phases in the range of 120–440°C within NBCs based on sulfuric-acid hydrolyzed cellulose nanocrystal (CNC) extracted from sugarcane bagasse (Figure 17). The glycosyl units of cellulose can undergo degradation due to decarboxylation, depolymerization, and decomposition. It is reported that the highly sulfated amorphous domains are more sensitive to low-temperature degradation compared to non-sulfated crystalline domains, which are more sensitive to higher temperature decomposition. This impacts the activation energies of the degradation process. The negatively sulfated groups contributed to the decreased thermal stability introduced on the outer surface of cellulose nanocrystal during the sulfuric acid hydrolysis [65].

Figure 17.

TGA/DTG curves of sulfuric-acid hydrolyzed cellulose nanocrystal (CNC) [65].

3.1.4 Thermal mechanical analysis (TMA)

Mechanical properties are very important for thin film evaluation, mainly in the ocular delivery of drugs, since it highly affects drug release rate, swelling, mechanical stability, and other properties [66, 67]. TMA measures the expansion and contraction of NBCs and the effect of crosslinking of the polymers or the enforcing materials [58]. TMA can be used to measure the coefficient of thermal expansion (CTE) of nanocomposite materials which indicates stiffness and energy losses as a function of temperature depending on the degree and the scale of dispersion of nanofillers within NBCs. It also allows the measurement of two different moduli of the nanocomposites, the storage modulus (E′), which is related to the ability of the material to return or store mechanical energy, and the loss modulus (E″), which is related to the ability of the material to dissipate energy as a function of temperature. DMTA data generally showed significant improvements in the storage modulus over a wide temperature range for a large number of polymer nanocomposites [58]. Figure 18 shows the use of DTMA to evaluate the storage of pectin/cellulose nanocrystal nanocomposite films with varying compositions of (NCC) [68].

Figure 18.

DMTA curves with Storage modulus (E′) for the neat pectin film and pectin/ CNC20, pectin/CNC50, and pectin/CNC80 nanocomposite films with 8% filler. (a) [68].

3.1.5 Integrated thermal analysis techniques

The integration of thermal analyzers with the microscopy allows visual monitoring of the solid transitions thus, capturing solid-state changes as a function of temperatures and time. For example, Hot Stage Microscopy (HSM) is a combined microscopical technique with DSC/TGA [59]. It has the advantage of evaluating sample morphology, solid state (amorphous, crystalline, polymorphism) transitions, desolvation, and miscibility. Figure 19 shows the optical micrographs of cellulose diphenyl phosphate at different temperatures. It can be seen that the product started to soften up at 150°C with a complete softening at 170°C [62]. Additionally, HSM can be combined with FTIR and or scanning electron microscopy enabling detailed information about the solid state of NBCs [59].

Figure 19.

Optical micrographs of C-A–Dp (sample M5 DSP = 1.19, DSA = 1.53) at different temperatures [62].

Additionally, gas evolving during the heating process can be trapped and analyzed using Gas chromatography–mass spectroscopy (GC–MS) techniques. These gases can also be chemically characterized using FT-IR and mass spectroscopy. Additionally, it is possible to combine AFM with DSC, TGA, or any other thermal analyzer enabling the evaluation NBCs [61].

3.2 Morphological properties using microscopy

The size and surface characteristics of NBCs can be acquired by microscopic methods. Optical detection and spectroscopy of a single nano-object can be achieved via detection of NBC interaction with a light beam, i.e., its elastic or inelastic scattering or absorption, or nonlinear ones (such as hyper-Rayleigh scattering or four-wave mixing) [69]. Surface roughness and/ or porousness, homogeneity, diameter, etc., are highly affected by solvent separation, for example, by evaporation resulting in shrinkage and wrinkle formation. Polarized Light Microscopy (POM) is primarily used in NBCs hydrogel evaluation due to their modest sample preparation steps. Craciun et al. investigated the chitosan self-healing hydrogels, designed as carriers for local drug delivery by parenteral administration. Based on the formation of an imine between chitosan and pyridoxal 5-phosphate, the active form of vitamin B6. POM images showed an intense birefringence in a sample of chitosan/pyridoxal 5-phosphate hydrogel, indicating the signature of an ordering degree (Figure 20). By coupling X-ray and POM data, it can detect the intermolecular forces that directed a supramolecular arrangement of the imino-chitosan chains [40].

Figure 20.

(A) Representative POM image of xerogel 1.5 t (to be representative, the hydrogels given in the figure have a different molar ratio of glucosamine/aldehyde units and/or different water volumes [40]. (B) Transmission electron microscope (TEM) images of the modified xanthan gum. Nanoparticles [14].

High-resolution methods are now used to get precise dimensions of the NBCs; in addition, they can be used to assess changes over time with regard to agglomeration, swellability, and shear-induced configuration. etc. [29, 70]. NBCs morphology using scattering techniques includes polarized and depolarized light scattering (DLS and DDLS, respectively). Electron microscopy is a technique with a nanometer scale resolution and is capable of imaging NBCs including Transmission Electron Microscope(TEM), Scanning Electron Microscope(SEM), and Atomic Force Microscopy (AFM). Electron microscopy enables the direct observation of the dimensions (i.e., length and width) of a given particle [71].

3.2.1 Transmission electron microscope (TEM)

TEM images provide good nanometric (and often subnanometric) resolution, allowing rapid screening of a large population of particles, thus avoiding major sampling issues. The method involves using a high-energy electron beam to bombard the sample. However, as TEM images are projections of the objects along the incident beam direction, it may be difficult to accurately measure the particle thickness. Depending on the amount of energy that was absorbed by the sample, the intensity of the beam that hits the viewing screen varies, and an image is made [71, 72].

In order to get an accurate measurement using TEM, the sample should be extremely thin (thickness below well below 1 μm) and well dispersed (no agglomerate) to allow transmission of electrons; additionally, the atomic number, density of the observed material, and on the energy of the incident electrons should be considered. Usually, this can be achieved using sectioning techniques for solid samples or preparation of NBCs using dilute suspensions (dispersion) [71]. Then, the sample is deposited on thin circular metallic grids (copper, carbon, etc.) with typical meshes around a few 10 of micrometers and accurately mounted in the sample holder for microscopy. Caution should be taken to avoid degradation of the copper grids when a high or low pH dispersion is investigated. Moreover, to avoid aqueous sample accumulation due to the hydrophobicity of the grids, glow discharge should be performed by placing the carbon-coated grids inside a partly evacuated chamber connected to a power supply. This allows the electron potential to ionize the gas within the chamber where negatively charged ions deposit on the carbon, giving the carbon film an overall hydrophilic (water-attracting) surface [71, 72]. Figure 20(B) depicts TEM image of modified xanthan-gum based [14].

NBCs main components, carbon, oxygen, nitrogen, and hydrogen, are not very dense, so sample staining is needed since the number of electrons they absorb is minimal compared to the intensity of the electron beam. Therefore, a heavy metal salt that readily absorbs electrons like lead, tungsten, molybdenum, vanadium, or depleted uranium is usually used. After staining, the sample is blotted, air dried, and ready to be examined in the microscope [71].

3.2.2 Scanning electron microscope (SEM)

SEM analysis technique uses electron selective detection methods capable of nano-resolution and chemical characterization of NBCs [70]. SEM micrographs can be used to study NBC porosity, the uniformity of pores, pore interconnectivity, and their size. NBC porosity can be a measure of the ability of NBCs to swell and deswell, which highly affects drug release mechanisms. Figure 21 depicts the uniform porousity of hydrogel NBCs based on chitosan (CS), xanthan gum (XG), monomer 2-acrylamido-2-methylpropane sulfonic acid (AMPS) that was successfully used to deliver acyclovir [73]. Pore dimensions are highly affected by the water content and the degree of crosslinking within the NBC. Crosslinking highly affects intermolecular physical connections among the chitosan. SEM can provide a detailed histogram describing the change in the number of pores with water content to enable optimization of the dispersion volume of NBCs, as depicted in Figure 22 [40].

Figure 21.

SEM images of acyclovir loaded hydrogel (A) At 200 μm (B) At 100 μm [73].

Figure 22.

SEM images for representative xerogels (scale bar: 50 μm) and corresponding histograms (to be representative, the samples given in the figure have a different molar ratio of glucosamine/aldehyde units and/or different water volumes) [40].

3.2.3 Integrated SEM techniques

SEM- Energy Dispersive X-ray Analysis (EDAX) can be used to study the morphological appearance of the hydrogels. Stanescu et al. Used SEM-EDAX integrated with elemental analysis to detect the proportions of elements like carbon and oxygen in chitosan and bacterial cellulose NBCs for wound dressing (Figure 23) [29].

Figure 23.

SEM images of: (a,c)BC/chitosan membrane surface; (b,d); lateral view of BC/chitosan; (e); scattering distribution crude chitosan surface, (f) EDAX elemental analysis [29].

3.2.4 Field emission gun scanning electron microscopy (FEG-SEM)

FE-SEM can be used to study the distribution and cross sections of the nanocomposites within a matrix [74, 75]. Niroomand et al. used FESEM to study the morphology of the cellulosic matrix and NBCs films [18]. Figure 24 depicts FE-SEM of differences in sample preparation of dried BC nanofiber, or NBCs film prepared by blender (Figure 24A) or homogenizer (Figure 24B) [18]. The film was coated with gold to minimize the electron charge (Figure 24C) [18].

Figure 24.

FESEM morphology of bacterial cellulose nanofibers after treatment with (A) blender and (B) homogenizer [76]. (C) FE-SEM image of the synthesized Nano-CS particles [18].

Figure 25.

AFM images of the NBC film surfaces are shown as a three-dimensional structure with their root mean square (RMS) roughness value: a cellulose film; b (2%), c (5), and d (15%) cellulose film containing 2, 5, and 15% nanochitosan particles, respectively [18].

3.2.5 Atomic force microscopy (AFM)

AFM provides information on morphology, surface topography (roughness and transparency), mechanical properties, and adhesion of NBCs. It also provides accurate measurement of NBCs size and size distribution. The high spatial and force resolution of AFM up to sub-nanometer scale and pico newton forces offers several unique advantages. It can also provide high-resolution mechanical testing along with images [70, 72]. Additionally, it also works with some special in-situ settings, such as imaging samples with fluid layers or wet samples. However, due to the scanning mechanism of AFM, an artifact may be introduced by contamination layers, temperature change, surface shear, or complex surface topography. The AFM scanning in high resolution also requires very small scanning steps. Along with the distance limitation of non-contact or tapping mode, the scanning of relatively large non-smooth areas or complex overall shapes can be very challenging [70]. Niroomand et al. reported the use of AFM to monitor the effect of nanochitosan addition on cellulose NBCs As can be seen in Figures 25 and 26. The 3-D AFM overviews showed a decrease in transparency and roughness of the cellulose-based NBCs when dosages of nanochitosan were increased, compared to the addition of 15% nanochitosan, which resulted in a rougher surface [18].

Figure 26.

AFM image of the nanochitosan particles and the histogram demonstrating the average size and size distribution of the nanochitosan particles [18].

3.3 Hydrophilicity of nanobiocomposite

Hydrophilicity is highly dependent on the NBC morphology, surface chemistry, water absorption, solid state, and porousity of an NBC. It greatly affects the wettability and dispersibility of NBCs and adhesion to the mucous membranes, and ability to adjust to curves and uneven textures like skin [18]. It is usually determined by monitoring the contact angle (CA)of a water droplet on a surface on NBCs film or matrix using a goniometer or a contact angle analyzer. The spreading out of a water droplet on an NBC surface indicates its hydrophilicity, while the resistance to spreading indicates hydrophobicity [77]. A low CA (below 90) between the droplet and the surface indicates hydrophilicity and wettability [18].

It has been reported that polysaccharides like chitosan and alginate are highly wettable; however, they have low mechanical strength. Their ability to absorb water is due to the presence of hydrophilic groups on the surface of their relevant NBCS. Thus, NBCs of these polymers usually have a hydrophilic nature. Once functionalized, the polymer will change the NBCs wettability depending on the functional group exposed to the surface. Additionally, the presence of a more compact microstructure of the nanocomposites due to the strong interaction within a polymer or the NBC will affect its wettability. It is important that surface hydrophilic group are free to interact with water in order to enhance wettability and is not fully engaged. Moreover, the presence of an amorphous state in an NBC allows the free movability of hydrophilic groups to interact with water, hence enhancing its water absorption and wettability [18].

Espino-Pérez et al. reported the increased hydrophilicity on NBCs based on cellulose nanocrystals (CNC) surface following esterification with phenylacetic acid and hydrocinnamic acid [52]. Similarly, Chiaoprakobkij et al. evaluated the water contact angle of films based on mechanically-disintegrated bacterial cellulose, alginate, and gelatin (BCAGG), plasticized with glycerol before and after curcumin loading (BCAGG-C). The study showed an increase in the angle with increasing curcumin content, as shown in Figure 27 4A–D. The water contact angle of the drug-free NBCs was 49.5, while after loading with curcumin concentrations (2, 4, 6, and 8 mg/mL), the contact angle ranged 54.7–73.3, indicating the hydrophilic nature of the drug-free NBCs [78].

Figure 27.

Water contact angles of NBCs based on bacterial cellulose, alginate, gelatin: (A) 2 mg/mL; (B), 4 mg/mL; (C), 6 mg/mL; and, 8 mg/mL (D)), solutions of curcumin at concentrations of 2, 4, 6 and 8 mg/mL [78].

3.4 Particle size distribution and determination and surface charge

NPs size plays an important role in cellular uptake and fate of the NPs within the body, consequently it affects the drug’s half-life and therapeutic efficacy. In addition, the particle charge (zeta-potential) has a large impact on surface recognition, surface interaction with biomolecules, and cellular targeting. Importantly, size and zeta-potential contribute significantly to the NP storage stability [79, 80]. The need for a monodisperse NP dispersion mandate the use of very advanced and facile methods that facilitate monitoring of NP size over time and following exposure to changes in temperature and pHs. The most recent methods in NPs size determination are dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE) [80, 81].

Visually, colloidal samples should be slightly hazy with a homogenous appearance termed the Tyndall effect. Figure 28 depicts NPs based on chitosan succinate 0.1% NPs crosslinked with polyphosphate and dispersed in 4.8 mM HCl [75]. In a colloid, the light scatters in different directions due to the colliding dispersed particles. It occurs when the diameter of an NP is in the range of roughly 40 to 900 nm, i.e., somewhat below or near the wavelengths of visible light (400–750 nm). Light scattering will occur when the diameter of dispersed particles is much smaller than the wavelength of light used [7, 75].

Figure 28.

Responses of ionotropic chitosan succinyl amide NPs (CS-H-80-TPP/EDC) to variable CaCl₂ concentrations [7].

DLS technique is based on exposing NP dispersion to a monochromatic coherent laser beam where the particle will move as a result of Brownian motion. With continuous collision between the particles, the distance between them will also change and hence fluctuations of the phase relations of the scattered light will be detected [82, 83]. Since the particle has different diameters, the number of particles within the scattering volume will vary in sedimentation with time. By observing the change in light intensity, this will be digitally correlated by photon analysis [84]. The DLS system measures the rate of intensity fluctuations and then uses this to calculate the size of the particles as defined in the Stokes-Einstein equation (Eq. (3) [81, 82]:

$$D=\frac{\overline{R}T}{\mathit{NA}}\frac{1}{6\mathit{\pi \eta }r}$$E3

where, D is the diffusion constant; $\overline{R}$ is the gas constant; T is the absolute temperature; η is the dynamic viscosity; r is the radius of the spherical particle; NA is Avogadro’s Number. Depending on the dispersity of the sample particle, light intensity and its decay will be correlated to give an idea about the size distribution of the sample NPs. The quality of measurement depends on the sample and the measuring device (the laser source, the detector precision, the correlator software, etc.) [80, 81].

It should be emphasized that several factors contribute to the accuracy of measurements. For example, a high sample concentration will affect the length of the path, allowing for the particle collisions resulting in a small path; hence, multiple collisions interfere with the measurement. Simple DLS instruments can measure at a fixed angle to determine the mean particle size in a limited size range. Since NBCs are mostly characterized by polydispersity, the light scattering by large particles can overwhelm the one by smaller particles. A multi-angle instrument that allows full particle size distribution is needed for accurate readings [80, 81].

In order to measure the size using this technique, the sample should be dispersed in the correct solvent (known refractive index, RI) at the optimum concentration and proper dilution. DLS measurement for dry samples can be achieved following dispersion in the proper solvent, as described before. The most commonly reported dispersion media is double deionized water (RI 193.39 nm at 25C) for pharmaceutical applications. In addition, glycerol and ethanol were also used with diluted concentrations [81]. Other measurements were done using methanol and toluene. The stability of the sample in the solvent is crucial, i.e. the particles should remain dispensed (undissolved with no aggregation) during the measurement. The purity of the dispersed particle and solvent and the exclusion of fiber and dust are highly needed prior to applying this technique appropriately [81, 82]. Figure 29 depicts an example of the particle size distribution of DLS investigation with NP dimensional distribution: NP from 0.5% PNIPAM/PVA concentration with various methyl oleate concentrations (top); nanoparticles from 5% PNIPAM/PVA concentration with various methyl oleate concentrations (bottom) [29].

Figure 29.

DLS investigation with nanoparticles’ dimensional distribution: nanoparticles from 0.5% PNIPAM/PVA concentration with various methyl oleate concentrations (top); nanoparticles from 5% PNIPAM/PVA concentration with various methyl oleate concentrations (bottom) [29].

Published data related to NBC size characterization include but are not limited to grafted pectin [13], chitosan phthalate and phenyl succinate [7, 79], thiolated chitosan [5], chitosan [30, 85], cellulose [86] and many others.

3.4.1 NBC surface charge (Zeta potential) measurement

The zeta potential is a measure of the electric charge at the surface of NPs, being an indirect assessment of their physical stability. The surface properties of NBCs will greatly impact the affecting the release properties and the interaction between the drug delivery system and the cellular receptor. The presence of specific chemical groups on the surface of the NBCs will also enable further functionalization by antibodies or by enzymes [41]. These properties greatly affect drug targeting resulting in site-specific drug delivery, for example, in tumor treatment [46].

Laser Doppler electrophoresis (LDE) involves measuring the change in the light scattering intensity due to the shift in the frequency of the wave as a result of interaction between a particle surface charge and the electric field. The direction and velocity of the motion are a function of particle charge, the suspending medium, and the electric field strength. Particle velocity is then measured by observing the Doppler shift in the scattered light. The particle velocity is proportional to the electrical potential of the particle at the shear plane, which is zeta potential [80].

When NPs are dispersed in water, they will gain electric charges. As a consequence, a concentration of oppositely charged ions (counterions) builds up at the particle surface. If these counterions are separated from or sheared off the particle by electrophoresis, a streaming potential can be measured in mV (Figure 30B). The measurement usually involves converting the electrophoretic mobility by the equipment software into zeta potential data through Smoluchwski’s approximation [24]. When performing the zeta potential measurement, it is important that the pH and the temperature are controlled with continuous dispersion of the NBC [83]. A negative charge in an NBC surface favors the stability and prevents aggregation (self-accumulation) of NPs. Zhou et al. reported enhanced stability of negatively charged sinapic acid-grafted-chitosan NPs loaded with black rice anthocyanins when compared to chitosan NPs alone [39]. Wang et al. reported the presence of a negative surface charge following grafting succinyl chitosan, which can be attributed to the ionizable –COOH group present at the NP surface at pH 7.4 [41]. Similar observations were reported by Chang et al. while monitoring the zeta potential change with pH in starch-modified NPs [56] (Figure 30B).

Figure 30.

(A) ZETA potential of PVA, PNIPAM, and PNIPAM/PVA/MO nanoparticles (a); Schematic representation of nanoparticles core-shell structure (b) [29]. (B) Effect of initial solution pH on zeta potential of amphoteric SNPs (CM-SNPs- C-2), CMSNPs, and CSNPs-2 [56].

Many published data have reported the use of zeta potential in NBCs characterization. The following are examples: cellulose NBCs [86], glucose-conjugated chitosan [48], starch [44], pectin [43], chitosan [7, 46], lecithin/chitosan phthalate and Phenyl succinate [79], etc.

3.5 Adsorptive properties

Surface adsorptive properties of NBCs, have a significant impact on its application as a drug delivery system, for example, for surface drug loading, functionalization by ligands (antigens, antibodies, etc.), enzyme immobilization, etc. [87, 88]. Additionally, It affects NBCs recognition, phagocytosis, elimination by macrophages, which can then further affect their transport and fate in the body [3, 15, 89].

Depending on the type of biocomposite, adsorption is highly affected by pH, ionic strength, adsorbent concentration, contact time, and temperature. Adsorption experiments can usually be conducted by shaking the NBCs or the polymer with variable concentrations of an adsorbent in a suitable container for a proposed time interval. The solutions are usually agitated at a constant speed in a temperature-controlled water bath at different temperatures for the required period. At a predetermined interval, samples are withdrawn, centrifuged, and the concentration of the adsorbent is analyzed using a suitable quantifying technique like UV [90, 91].

Natural polymers like chitosan have –NH₂ and –OH adjacent to –NH₂ in its backbone, which enhances its adsorption properties for many metal ions such as aluminum, silver, zinc, etc. [90]. Lee et al. investigated the adsorption of mucin to chitosan (mucoadhesiveness) during evaluating thiolated CS Intranasal delivery of theophylline [92] Similarly, the hydroxyl groups on the surface of nanocellulose(cellulose nanofibrils and cellulose nanocrystals) allowed electrostatic adsorption, making them suitable for enzyme/protein immobilization [93].

Adsorption evaluation generally involves gentle mixing of a precise volume of NBCs dispersion with a fixed volume of drug, enzyme, or metal solution, etc., with continuous shaking at a specific speed in a thermostat-regulated shaker at 25°C. Once equilibration is attained (e.g., 20 h), the mixture is filtered, and the concentration of residual adsorbent (enzyme, etc.) in the filtrate is determined by UV–VIS spectrophotometry. Oshima et al. reported this method in measuring the adsorption of protein (lysozyme) to the surface of phosphorylated cellulose using Eq. (4) [91]:

$$\%\mathit{\text{Adsorption}}=\frac{\mathit{Co}-\mathit{Ce}}{\mathit{Co}}\times 100\%$$E4

$$\mathit{\text{Amountadsorbed}}\left(q\right)\left[\mathit{\text{umol}}/\mathit{gm}\right]=\frac{\mathit{Co}-\mathit{Ce}}{W}\times \mathit{V}$$

Where C₀ and C_e are the protein concentrations before and after adsorption in mol/ml, W is the dry mass of adsorbent in gm, and V is the volume of solution in ml. Adsorption isotherms of enzymes like lysozyme can be obtained at constant temperature (for example, 30°C) using aqueous solutions containing varying concentrations of lysozyme and adsorbents [91].

The effect of molecular weight on NBCs has a great role in adsorption properties. Riegger et al. investigated the impact of molecular weight of six commercially available, highly deacetylated chitosan based NP on the adsorption of diclofenac and carbamazepine. They reported an adsorption capacity of up to 351.8 mg/g diclofenac for low MW chitosan NPs, and all chitosan NPs showed superior adsorptions when compared to untreated chitosan. Hence, the results suggested the use of the prepared chitosan NPs as promising adsorbers for diclofenac and carbamazepine [85].

3.6 Gelling properties

Gel formation can be obtained using many natural polymers like alginate and chitosan, etc., which are capable of forming a 3Dimetnional structure upon crosslinking of their polymeric chains. Remarkably, a hydrogel is one type of gel that swells upon exposure to water, enabling its use in the delivery of many drugs [94]. The type and degree of crosslinking affect the nature of the gel, either tough or soft gel, the solubility, and its mechanical strength [4, 95]. There are two types of methods to prepare gels: physically (by a change in pH or temperature) and chemically (by electrostatic, covalent crosslinking, biological cell crosslinking, free radical polymerization, and click chemistry) [40, 66]. In general, ionic gelation favors mild conditions and results in soft gels (solvent, pH and temperate, etc.) [96]. Alginate has dominated among all hydrogels and is the most widely used hydrogel for encapsulation due to its low cost, high availability, and durability, as well as its nontoxicity to host organisms and well-established encapsulation process. When multivalent cations like Ca²⁺ are present in an aqueous solution, certain polymers like alginates and the like have the necessary characteristics to construct suitable matrices [11, 97]. Alginate has been successfully used to encapsulate cells [98].

On the other hand, covalent crosslinking involves harsh conditions (toxic materials like glutaraldehyde, high temperature, etc.) and favors more tough gels [29, 66]. Biocomposites like pectin/chitosan gel prepared by the casting method have been optimized by varying their components using lactic acid or glycerol as solvents. Also, an antibacterial test against Bacillus subtilis confirms that the pectin and chitosan retains their antibacterial property in biocomposite materials [99].

3.6.1 Visual, optical transparency (clarity) and surface evaluation of hydrogels

The clarity and surface smoothness of gels mainly depends on the presence and structure of insoluble components [100]. The transparency of a gel indicates the solubility of the components and homogeneity of the fabrication process. Direct evaluation of visual transparency is performed by the eye, while the optical transparency of the formulated hydrogel is analyzed by a UV as described previously [101]. 100% light transmittance in distilled water indicates the optical transparency of a hydrogel. It is important that both of these measurements are performed at different temperatures, for example, 25 and 37°C, accompanied by pH measurement. Additionally, SEM analysis is usually performed to evaluate the smoothness, homogeneity, or heterogeneity of the gel surface [66].

3.6.2 Sol–gel transition behavior, gelation time, and gelation temperature

A sol–gel transition occurs through additional intermolecular interactions of a hydrophobic nature, leading to the formation of a turbid gel that can be achieved by exposure to variable temperature, pH, or shaking. It must be determined at a physiological temperature 37°C) since it will greatly affect the drug release, injectability and storage conditions [96]. The gelation temperature is usually measured by placing the polymer solution in a glass vial and exposing it to heat with gentle shaking. The content is observed for gelation on intervals while inverting the glass vial at a 90° angle for 1 min. Once flowability is stopped, the temperature is recorded as a gelation temperature. The time needed for a solution to stop flowing (gel formation) is termed gelation time [96, 102].

3.6.3 Sol–gel fraction

Sol–gel fraction examination is carried out to determine the sol and gel fraction in any prepared hydrogel. The liquid portion of the hydrogel is expressed as the sol fraction. In this test, discs of the dried hydrogel are weighed(W_i) and kept in boiling water at 100°C for approximately 4 h. After a certain time, the discs are removed from the water bath and dried at room temperature for 24 h or a low-temperature oven to a constant weight (W_d). For calculating the sol and gel fraction of the hydrogel, Eq. (5) is employed [103]:

$$\mathit{GF}\%=\frac{\mathit{Wd}}{\mathit{Wi}}\times 100\%$$E5

$$\mathrm{s}=1-\mathrm{GF}$$

where s is the sol fraction, GF is the gel fraction.

3.6.4 Water absorption capacity (WAC)/fluid uptake ability

This test usually involves immersing an accurately weighed film or hydrogel in water or suitable fluid at room temperature and allowing it to equilibrate. Following a specific time (6 and 24 h), the sample is removed, and the surface water fluid is removed (wiped) gently and re-weighed. Water content /fluid uptake (%WAS) is usually determined by a precise balance and calculated using the Eq. (6) [103]:

$$\%\mathit{WAC}=\frac{\mathit{Ws}-\mathit{Wd}}{\mathit{Wd}}\times 100\%$$E6

where Wd and Ws are the weights of the dry sample and wet sample, respectively. A similar procedure is applied in measuring the Fluid uptake ability by immersing the weighed samples in PBS (pH 7.4) and artificial saliva (pH 6.2) at 37°C.

Additional tests to characterize NBCs-based gels include:

3.6.5 Surface wettability

This can be evaluated as a static water contact angle by monitoring a water droplet from different locations using a contact angle analyzer [103].

3.6.6 Hydrogel oxygen and water permeability

The oxygen transmission rate (OTR) of the films is needed for dermal dressing to ensure the non-occlusiveness of hydrogel/films. Usually, it is determined by an oxygen permeation analyzer at 25°C and 0% relative humidity. In this test, one side of the sample is exposed to a nitrogen atmosphere, while the other side is exposed to an oxygen atmosphere. When the concentration of oxygen on the nitrogen side becomes constant, the test is considered complete [104].

On the other hand, water vapor permeation is measured by a water permeability analyzer. The pre-weighed sample is placed in a test dish containing a desiccant, and the assembly is placed in a controlled atmosphere at 37°C and 98% relative humidity. Periodic weighting is performed to determine the rate of water vapor movement through the specimen into the desiccant and plotted against time [103].

3.7 Mechanical properties

The need to measure the mechanical properties (elasticity and flexibility) is mandatory in formulations applied directly to the skin or the tissues. NBCs hydrogels are flexible, porous and can be fabricated by chemical or physical crosslinking nanomaterials as described previously. Varying the conditions of the crosslinking process (crosslinker type, time, temperature, etc.) can be used to achieve a strong, flexible hydrogel [105].

Among the important mechanical properties are tensile strength (TS) and elongation at break (EB), which are measured using a tensile strength tester. TS and EB are usually calculated using the Eq. (7) [106]:

$$\mathrm{TS}=\text{Maximum force}:\left(\text{Film thickness}\right)\times \left(\text{Film width}\right)$$E7

$$\mathit{EB}=\frac{\mathit{\Delta L}\times L0}{100}$$

Where ΔL and L0 are the elongation of the specimen at the moment of break, and the initial length of the specimen, respectively.

The effect of additives on the mechanical strength of NBCs has been studied by Kassab et al. They investigated their mechanical reinforcement capability for k-carrageenan biopolymer on cellulose nanocrystals (CNC). The obtained CNC was dispersed into a k-carrageenan biopolymer matrix at various CNC contents (1, 3, 5, and 8 wt%), and the prepared films were further characterized. The incorporation of CNC enhanced the mechanical properties compared with the neat k-carrageenan (k-CA) film, as seen in Figure 31. All nanocomposite films have higher tensile strength compared to films based on neat k-CA biopolymer. This is attributed to the great improvement attained by the addition of CNC. Furthermore, the researchers reported an increase in the modulus and strength by increasing the CNC content from 1 to 8 wt%, with slight variation in the toughness of CNC of the biocomposite [65].

Figure 31.

Typical stress–strain curves of neat k-carrageenan (k-CA) film and its nanocomposites at different CNC contents (1–8 wt%) [65].

Chaichi et al. implemented a statistical optimization approach to study the effect of additives like Ca²⁺ as a crosslinker and glycerol on the tensile strength of NBCs. The researchers demonstrated that Ca²⁺ ions could significantly reduce the swelling and elongation to break while increasing the tensile strength of the NBCs [106]. In a similar study, Chiaoprakobkij et al. reported curcumin-loaded film’s formulation based on bacterial cellulose/alginate/gelatin using mechanical and casting methods. Films were stretchable with the appropriate stiffness and enduring deformation, which enabled dermal application when sufficiently hydrated. Additionally, the films have good mucoadhesive properties, which enhance the antibacterial activity of curcumin against E. coli and S. aureus [78].

3.7.1 Tensile strength and elongation to break

The consistency among the polymer chains, flexibility (elongation before breakage), and ability to resist extension can be measured using tensile strength [106]. Adam et al. studied the effect of incorporating gum Arabic κ-carrageenan biocomposite in hydroxypropyl methylcellulose (HPMC) hard capsules shell. The optimization involved the use of variable ratios of the hard capsule constituents in order to achieve a capsule with good tensile strength and optimum disintegration time. The researchers suggested that this biocomposite can be an alternative to ordinary gelatin used in capsule shell formulation [107].

3.7.2 Stiffness of the material

Kurowiak et al. described the measurement of sodium alginate-based hydrogel when subjected to static tensile testing to determine its elasticity (Young’s modulus). The studies showed that hydrogel crosslinked with calcium ions showed a lower mechanical strength compared to the one crosslinked with Ba²⁺ cations (Figure 32). The researchers attributed the difference to the increased barium affinity to alginate monomers (G blocks), resulting in a hydrogel by forming the egg-box structure, characteristic of alginate NPs [108].

Figure 32.

Strain–strain analysis plot for the proposed sodium alginate-based material cross-linked with Ca²⁺ or Ba²⁺ cations using different cross-linking times after 72 h cross-linking time [108].

3.8 Rheological properties

Rheology is the study of how materials deform when a force (shear) is applied to them. Rheological properties affect fabrication conditions and the quality of the fabricated products. These materials are mostly liquids or liquid-like materials. Rheological measurements are also very useful for characterizing the flow properties of emulsion systems and predicting their behavior during manufacturing, storage, and drug administration [66, 109].

Basically, the rheological properties of NBCs are affected by the natural polymer properties and the additives included within the formula and the technique used in the formulation. The control of these two factors allows the optimization of the formula to suit the application in relation to the route of administration, for example, ocular, nasal, at the tumor site by injection, etc. Properties that can affect the rheology of NBCs include molecular weight (MW) and its distribution (MWD), morphological, molecular structure, and orientation under electric or magnetic fields. High molecular weight polymer exhibits thixotropic behavior with high resistance to extreme temperatures, freeze–thaw cycles, pHs, and salt concentrations [15]. Formulation factors include the presence of an additional compound or impurities (crosslinkers, surfactants, stabilizer, etc.), the solvent used, ionic strength, pH, NBCs concentration, pressure, and temperature [71]. It should be noted that in strongly crosslinked samples, no rheological measurements could be performed due to their brittle properties. Sample assessment includes carful control of temperature and prevention of the solvent evaporation [66, 71].

Rheometers can be divided into two categories: rotational and capillary types. Two major types of rheological experiments can be performed utilizing parallel-plate or rotational rheometer, the sweep tests (varying strain, frequency, and temperature) and the steady shear sweeps (Figure 33) [110]. The principle of each test and examples are described in the following paragraphs.

Figure 33.

(A)Time sweep, (B) Strain sweep, (C)Temperature sweep, (D) Frequency sweep, and (E)Creep Compliance, (F)Creep Recovery [95].

3.8.1 Flow curves (steady shear flow)

The importance of having a consistent viscosity during storage is a vital feature of drug delivery systems. Flow curves describe the rheological behavior of a material, more specifically, the dependency of the viscosity on the applied shear rate and the tendency of a material to flow. The plot is represented by viscosity as a function of shear rate (log relationship can be used). These are usually used to evaluate the viscosity of hydrogels using different crosslinking ratios. Formulation of NBCs in the nanoscale can increase the viscosity. Ahmad et al. reported that the viscosity of starch-based NPs dispersion was influenced by the shape, size, and distribution of the starch granules and also by the amylose content with variable viscosity depending on the source of starch. As can be shown in Figure 34, a decrease in the size of starch at the nanoscale increased the viscosity of the dispersion compared to the native starch [44].

Figure 34.

Flow curves (steady shear flow) for native and nano starch particles against shear rate.: Horse chestnut particle (HSP), Water chestnut Particle (WSP), Lotus stem particles (LSP), Horse chestnut (HS), Water chestnut (WS), and Lotus stem (LS) [44].

Mishra et al. reported improved shear stability of the polyacrylamide-grafted pectin hydrogel compared to the pectin-based hydrogel. The viscosity of the polymer solutions decreases with an increase in shear rate. Both the aqueous 5% solutions of grafted Pectin and Pectin showed strong pseudoplastic behavior. As can be seen in Figure 35, at low and high shear rates, the viscosity of the grafted pectin solution was higher than the pectin solution. This suggests that grafted pectin solution was more shear stable than the ungrafted pectin, which can be attributed to longer branches in the grafted pectin [49].

Figure 35.

Viscosity versus shear rate curve of 5% grafted pectin solution(A); Viscosity versus shear rate curve of 5% pectin solution(B) [49].

In addition, studying the viscosity dependence on the shear rate enables the classification of the hydrogel into those which exhibit thixotropy or its opposite phenomenon, rheopexy behavior. In this test, the sample is exposed to an increased shear rate, and the viscosity of the hydrogel decreases up to a certain minimum which indicates thixotropic behavior. After this, the shear rate is reduced, which leads to increased values in the viscosity, which are higher than the original viscosity values for the respective shear rate. This phenomenon is known as negative thixotropy or rheopexy.

3.8.2 Time sweep test

This test evaluates structural changes for a specific material after applying a shear over a certain time. These changes can be observed following evaporation of the solvent, curing, gelation, polymer degradation, or recovery. For example, the gelation time can be related to the kinetics of the gelation reaction, which is defined as the crossover point of the storage (G′) and loss (G″) modulus [95]. It should be emphasized that no rheological measurements could be performed for strongly crosslinked samples due to their brittle properties [111]. Stanescu et al. reported that the uncrosslinked samples of bacterial cellulose (BC)/chitosan NBCs loaded with silver sulfadiazine showed the lowest shear viscosity values compared to crosslinked NBCS (Figure 36A) which, can be attributed to network development during crosslinking process. The prolonged exposure to the crosslinker resulted in a higher shear viscosity. Additionally, the presence of BC reduces CS shear viscosity when compared to CS alone (Figure 36B), which showed higher shear viscosity values [29].

Figure 36.

Rheological behavior (shear viscosity) measurements for (A) Bacterial Cellulose (BC)/chitosan and (B) chitosan alone [29].

Strain sweep test (amplitude sweep).

This test is used to characterize hydrogels using increasing oscillatory strain at a constant frequency on the storage (G′) and loss (G″) modulus of the hydrogel to determine the linear viscoelastic region (LVR) (Figure 37A) Jannatamani et al. evaluated nano-hydrogels and films based on the wood Cellulose NanoFibers (WCNF), Bacterial Cellulose NanoFibers (BCNF), and Chitin NanoFibers (ChNF LVR). At low shear stress, the moduli are independent of the increasing stress. However, as the stress is increased, the G′-G″ crossover point potentially reaches, at which the gel–sol transformation occurs, and the material starts to behave like a fluid. Additionally, a Strain sweep in hydrogels can be used to estimate the threshold strain required above which shear thinning behavior is observed (Figure 37B) [112].

Figure 37.

Storage and loss modulus of 0.5 wt% (A) and 1 wt% (B) concentration of WCNF, BCNF, and ChNF nano-hydrogels as a function of strain [112].

3.8.3 Temperature sweep

Sometimes it is termed the temperature ramp test, which enables predicting the structure of the hydrogel, and its stability when subjected to a certain range of temperatures. This can be achieved by studying the storage (G′) and loss (G″) modulus in a certain temperature range with an evaluation of the sol–gel transition of the hydrogel (Figure 33C). The point at which the viscosity drops suggests the temperature above which the hydrogel starts to degrade or de-crosslink. Plotting the viscosity vs. temperature will enable predicting the temperature when a hydrogel will degrade or uncrosslinked [95].

3.8.4 Frequency sweep

The effect of additives on viscoelastic properties of hydrogel can be studied by varying the frequency and evaluating its relationship with the storage (G′) and loss (G″) modulus (Figure 38). Ajovalasit et al. used a frequency sweep test to evaluate the impact of additives like glutaraldehyde glycerol and PVA on the properties of Xyloglucan-based hydrogel films for wound dressing. They found that the addition of glycerol does not impact the rheological properties, whereas the addition of glutaraldehyde moves the G′ and G″ crossover point to lower frequencies. Interestingly, the addition of PVA decreases the storage and loss modulus values (G′ and G″) [113].

Figure 38.

Dynamic mechanical analysis in frequency sweep mode of precursor dispersions and films. Solid symbols: storage modulus; open symbols: loss modulus. (A) Aqueous XG and XG/ PVA before (XG_disp and XG-PVA_disp) and after addition of glycerol (XG(Gro 1)_disp and XG-PVA(Gro 1)_disp). (B) Precursor aqueous dispersions of the chemical films obtained by addition of glutaraldehyde in the same samples illustrated in panel (a) [113].

3.8.5 Creep compliance, creep recovery, and stress relaxation

This test is used to evaluate the elasticity of hydrogel films when a sample is subjected to a constant static load (strain) and how the structure recover following withdrawing this strain. In addition, it enables predicting hydrogel behavior following frequent use in real. Thus an increasing strain reaches an equilibrium after a certain time. After that, no further stress is applied, and the recovery of the sample is recorded over a certain fixed time [95].

Stress relaxation is the inverse of the creep compliance test, where a stress relaxation test subjects the sample to a constant strain and measures the stress exerted by the sample. It gives an idea of how well materials can dissipate stress over time at a constant strain. Craciun et al. evaluated the rheological properties of chitosan-based hydrogel compared to chitosan/ pyridoxal 5-phosphate (vitamin B6 precursor) based hydrogels used for local action, in tumors or on wounds. Chitosan (NH₂ source) and the aldehyde of pyridoxal (CHO) form hydrogel simultaneously due to imine group formation. The study revealed the dependence of gel formation on the ratio of NH₂/aldehyde ratio of chitosan /pyridoxal 5-phosphate (Figure 39). A higher recovery degree was achieved when the ratio was less than 3 [40].

Figure 39.

Effect of NH₂/aldehyde ratio on the structure recovery ability determined by the continuous step strain measurements with various dilutions and NH₂/CHO ratios [40].

4. Conclusion

Nanobiocomposites have been investigated in many fields, including the medical and pharmaceutical fields. For a successful application of NBCs as a drug delivery system, it is essential to perform an extensive physicochemical characterization of these NBCs. Each property discussed in this chapter has an extreme effect on the final appropriateness of these NBC as a drug delivery system. The impact of these properties can vary from drug-polymer interaction to therapeutic efficacy, safety, and stability. Therefore, a comprehensive characterization of each property will enhance achieving a safe and effective drug delivery system.