# All-aqueous liquid crystal emulsions for 4D laser-printing of dual-stimuli response porous 3D printlets with on-demand remote control of drug release

Mahdiyar Shahbazi, Henry Jäger, Rammile Ettelaie, Peyman Asghartabar

Kashi, Delphine Huc-Mathis, Jianshe Chen

► To cite this version:

Mahdiyar Shahbazi, Henry Jäger, Rammile Ettelaie, Peyman Asghartabar Kashi, Delphine Huc-Mathis, et al. All-aqueous liquid crystal emulsions for 4D laser-printing of dual-stimuli response porous 3D printlets with on-demand remote control of drug release. Chemical Engineering Journal, 2024, 502, pp.157614. 10.1016/j.cej.2024.157614. hal-05108505

HAL Id: hal-05108505

https://agroparistech.hal.science/hal-05108505v1

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# All-aqueous liquid crystal emulsions for 4D laser-printing of dual-stimuli response porous 3D printlets with on-demand remote control of drug release

Mahdiyar Shahbazi<sup>a,*</sup>, Henry Jäger<sup>a,*</sup>, Rammile Ettelaie<sup>b</sup>, Peyman Asghartabar Kashi<sup>c</sup>, Delphine Huc-Mathis<sup>d</sup>, Jianshe Chen<sup>e</sup>

## ARTICLE INFO

### Keywords:

- Laser 4D printing

- Liquid crystal

- Complex coacervation

- CNC

- Cholesteric self-assembly

- Thermoresponsive behavior

- polarized-LSCM

## ABSTRACT

The integration of responsive materials with 3D printing can lead to fabrication of functional smart 4D structures for use in health, biofabrication, and biosensing. Here, we utilized an all-aqueous two-phase system made from chitosan and carrageenan to create a bioactive all-water emulsion encapsulating curcumin to develop 4D printed 'printlets' for possible drug delivery. Specifically, we described the production of an original all-aqueous cholesteric bicontinuous liquid crystal (LC) emulsion, which was accomplished by homogenizing or sonicating a rod-like cellulose nanocrystal (CNC) with two thermodynamically immiscible, phase-separating carrageenan and chitosan dispersions. Stable water-in-water emulsions were effectively prepared by mixing or sonicating the respective CNC/biopolymer dispersions, resulting in micrometric CNC/chitosan dispersed droplets (loaded with curcumin) in a CNC/carrageenan continuous phase. The LC-based emulsion showed a confined 3D percolating bicontinuous network with cholesteric self-assembly of CNCs within the carrageenan phase, while the nano-particles in the chitosan phase remain isotropic. After laser 3D printing of the freeze-dried LC-based emulsion, 3D macroporous "printlets" encapsulated with curcumin were produced exhibiting a dual-stimuli-responsive to temperature and redox. The curcumin-loaded 3D 'printlets' exhibited superior thermosensitive properties and maintained a sustained drug release profile, exhibiting much greater on-demand release mode at human body temperature. Notably, the 3D porous "printlets" demonstrated a gated response to the aqueous environment, showcasing potential advancements in the realms of supramolecular sensors and drug delivery systems.

## 1. Introduction

Laser powder bed fusion, L-PBF (originally termed selective laser sintering) is a laser- or electron beam-based 3D printing, where a laser beam selectively fuses or sinters powder particles to construct an object layer-by-layer. This technique offers the capability to design and prototype for the production of personalized and custom-design 3D pharmaceuticals. The majority of the printing inks processed by 3D printing, especially L-PBF, are fundamentally static [1]. What is needed are printing precursors that can respond to changes in their environment, thereby altering the properties of the final inks to achieve the desired effects. This is then most anticipated regarding biofabrication to develop

implantable objects or in-vitro models [2], compliant soft robotics for healthcare [3], biotechnology manufacturing custom-made objects [4,5], drug delivery in pharmaceuticals and growth factors [6,7] and devices that work at the human-machine interface or for handling fragile objects [8]. Thus, the term "4D printing" originated and described the printing of 3D structures produced by 3D printing with stimulus-responsive properties (also named "smart materials"). As the responsivity and geometry of printing components could be altered, effective application is proposed for 4D structured functional materials. The development of four-dimensional (4D) printing methodology is predicated upon the foundation of three-dimensional (3D) printing, with the addition of a temporal dimension, thereby introducing a "space-time

<footnote><sup>a</sup> Institute of Food Technology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190 Vienna, Austria</footnote>

<footnote><sup>b</sup> Food Colloids and Bioprocessing Group, School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, UK</footnote>

<footnote><sup>c</sup> Faculty of Biosystem, College of Agricultural and Natural Resources, Tehran University, 31587-77871 Karaj, Iran</footnote>

<footnote><sup>d</sup> Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, 91120 Palaiseau, France</footnote>

<footnote><sup>e</sup> Food Oral Processing Laboratory, School of Food Science & Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: shahbazim00@yahoo.com, mahdiyar.shahbazi@boku.ac.at (M. Shahbazi), henry.jaeger@boku.ac.at (H. Jäger).</footnote>
axis" that is derived from the conventional 3D Cartesian coordinate system. This concept was initially proposed by Professor Skylar Tibbits at the Massachusetts Institute of Technology [9].

Various materials are used as printing inks for 4D printing, including polysiloxane thermosets, shape memory polymers, (anisotropic) hydrogels, and liquid crystalline (LC) materials, which can be processed through an assortment of 3D printing technologies. As a prime instance, hydrogels endow a high and reversible deformation according to swelling/deswelling phases, which are programmed to respond to many possible triggers [10]. However, they must be hydrated to function, which poses a problem in warm or dry surroundings. Compared to hydrogels, polysiloxane thermosets contain special nanofillers and are exposed to produce structures with reversible preprogrammed deformation in dry surroundings. However, they lack versatility concerning material functionalities [11]. Shape memory polymers are also a promising type of active polymers with numerous applications in a variety of 3D printing technologies, showing great potential for the 4D printing process. Unfortunately, shape memory polymers cannot mainly cycle between un-actuated/actuated phases separately (known as "one-way" shape memory), which requires reprogramming upon each actuation stroke.

LC-based materials merge the functional properties of all the mentioned compounds, which are partially ordered, anisotropic, reversible, and self-organized phases. These materials demonstrate tunability in terms of material functionalities in both wet and dry environments. As a mesophase dynamic self-assembled system, these types of materials show an exclusive combination of liquid fluidity and crystalline order, which can spontaneously organize directionally [12,13]. They are broadly utilized to manufacture reprogrammed structures, which can optically monitor evidence on their environment including chemical and biological stimuli, mechanical shear, and temperature variations [10–12]. There are two categories of LCs: thermotropic through temperature changes and lyotropic through concentration changes of a dispersant in an isotropic solvent [14]. Due to their exclusive optical, thermal, mechanical, and responsive properties, there has been huge attention to applying LCEs in a variety of biomedical applications [15,16], biotechnology [17], food [18], sensing [19], energy [20], water treatment [21], photonics [22], and soft actuation [23].

Water-in-water (W/W) emulsions, which contain droplets of one aqueous phase dispersed in another, represent a less common type of liquid/liquid biphasic system. The separation into two distinct phases in such systems is driven by the presence of hydrophilic molecules that are soluble in water but not compatible with each other with ultralow interfacial tension [10,11]. Emulsions that consist of at least one phase composed of an anisotropic fluid are referred to as "liquid crystal (LC) emulsions". LC emulsions have significant impacts on the structure of both water-in-oil (W/O) and oil-in-water (O/W) systems, leading to the formation of various complex assemblies due to anisotropic elasticity, interfacial interactions, elastic distortions, and topological defects. Anisotropic colloidal particles such as graphene oxide, viruses, and amyloid fibrils can generate aqueous lyotropic LCs at high concentrations. In the process of LC formation, microdomains of anisotropic droplets spontaneously nucleate from the isotropic suspension. To create a stable water-in-water (W/W) emulsion, hydrophilic polymers are introduced to induce repulsive forces between the two aqueous phases [10]. This emphasizes the importance of selecting appropriate polymers that can be effectively dispersed in the liquid crystal (LC) system, enabling the coexistence of LCs without compromising the integrity of the emulsion and LC order.

The acid hydrolyzing of natural cellulose commonly leads to the formation of rod-shaped cellulose nanocrystals (CNCs) with negative charges, which could develop LCs above their critical concentration [23]. These nanomaterials can also produce a variety of electrostatically stable LC structures, which relate to the concentration, surface charge, and size of the particles [24,25]. Isotropic, biphasic, and chiral nematic are typically three distinct LC phases for CNCs. When the crystals are

randomly orientated at a low level of CNC, the LCs occur as isotopic regions. Alternatively, the crystals orient and structure to a chiral nematic phase when a critical concentration is attained. Controlling and understanding the self-assembly process of LCs and the resulting helical arrangement of CNCs are crucial objectives. These objectives contribute to the development of the next innovative materials with desirable mechanical and optical properties [26]. In comparison to photonic crystals developed by the self-assembly of spherical colloidal particles without LC functionality, the helical self-assembly of rod-like CNC particles in a liquid crystalline state results in a more intricate internal structure. This complexity imparts chirality to the material and increases its sensitivity to circular polarization. Simultaneously, it significantly improves the functionality of water-in-water emulsions [27–29].

Several works attempt to establish an effective and straightforward method for stabilizing all-aqueous interfaces through the complexation of two oppositely charged polyelectrolytes [30–33]. For example, the dispersion made from the polycation chitosan and the polyanion cellulose nanocrystals results in an incompatible polyelectrolyte composite that is prone to coacervative phase separation. When an aqueous droplet containing one type of polyelectrolyte interacts with an external phase that contains the other polyelectrolytes, complexes are formed at the interface where these two polyelectrolytes converge. To ensure the droplets remain stable and to create reliable microcapsules, it is essential to adjust the flow of polyelectrolytes from both the droplet and external phases to ensure they converge in adequate amounts at the interface. The resulting polyelectrolytes/polyelectrolytes microcapsules showcase several advantageous properties, including ease of fabrication, stability, responsiveness to environmental conditions such as pH and salt levels, and the capacity to support living microorganisms. In this context, a nomenclature, polyelectrolytes/polyelectrolytes, where any membrane-forming components listed before the slash can be developed in the droplet phase, while those after the slash are incorporated into the external phase.

Currently, 4D printing involves the utilization of various function- alized inks with specific formulations and structural designs. Within this field, the transformation of shapes resulting from this ink combination through 3D printing techniques represents a significant research chal- lenge that needs to be tackled for the advancement of 4D printing. The thermoresponsive macroporous 3D structures exhibit potential as a drug delivery system, with an LCST transition occurring at body temperature. These structures can be utilized in the realm of intelligent release of bioactive compounds. Moreover, due to the amphiphilic properties of the 3D structure, it is possible to load both hydrophilic and hydrophobic medications into these objects. This enables the attainment of a sus- tained drug release process, thereby ensuring appropriate therapeutic efficacy.

In this study, an innovative W/W bicontinuous structured LC emul- sion was introduced featuring intraphase trapping of CNC with hierar- chical cholesteric self-assembly. Specifically, the utilization of the aqueous binary biopolymer systems consisting of carrageenan and chi- tosan as the primary components was outlined. Within this system, the dispersed CNC could maintain cholesteric ordering in the presence of incompatible biopolymers. In this case, the combination of self- assembled CNC particles with immiscible binary biopolymers to facili- tate the formation of an all-aqueous bicontinuous structured LC emul- sion was demonstrated. We also evaluated the manipulation of W/W emulsion morphology a bicontinuous structure to aspherical droplets through adjustments in the volume ratio of biopolymers, thereby regu- lating the liquid-liquid phase separation process. Our research also aims to encapsulate curcumin, a model hydrophobic drug, within a coacer- vate composed of chitosan and yield beneficial functionality. In this case, another goal of this study was to examine the potential of employing L-PBF as an innovative 3D printing method to fabricate innovative, self-supporting 3D/4D printlets from pharmaceutical-grade polymers. The final section aimed to explore the feasibility of these structures in customizing drug performance based on individual patient
needs. By incorporating this technology into pharmaceuticals, the 3D/ 4D printlets could be utilized to adjust the drug release properties of oral medications while preserving the formulation composition.

## 2. Experimental section

### 2.1. Materials and methods

CNC was sourced from Qihong Technology Co. (China) and stored at 4 °C to prevent drying and bacterial growth. The CNC concentration in the aqueous gel is approximately 10 % wt., and the crystallinity index is 75.6 %. Tetramethylrhodamine isothiocyanate (TRITC) and calcofluor white (CFW) stain were purchased from Sigma-Aldrich (Steinheim, Germany). Kappa-carrageenan (KC) was obtained from Tokyo Chemical Industries (Tokyo, Japan), with a viscosity of 10–25 mPa s and a concentration of 0.3 % in water at 25 °C. Chitosan (CS) was supplied by Sigma-Aldrich (Steinheim, Germany), with a molecular weight exceeding 75 % deacetylated. Fluorescein isothiocyanate (FITC) [34] and rhodamine B isothiocyanate (RBITC) [35] were purchased from Sigma-Aldrich (Steinheim, Germany). All chemicals were used as received without further purification.

#### 2.1.1. Preparation of CNC/CS and CNC/KC suspensions

To prepare the CNC suspension, 200 mL of Milli-Q water was added to concentrated CNC gel to achieve a uniform concentration of 5.0 % wt. The resulting mixture was then sonicated for 3 min in an ice bath to create a homogeneous dispersion. The sonicated mixture was transferred to a 300 mL bottle and allowed to stand for 48 h at room temperature. During this period, the mixture separated into two distinct phases: a clear upper isotropic phase and a lower cholesteric phase (data not shown). To prepare CNC/CS and CNC/KC suspensions, each biopolymer was dispersed in the CNC suspension (5.0 % wt.) followed by 2-min sonication. Regarding CNC/CS, curcumin (0.025 % wt.) and dilute acetic acid (1 % wt.) were also introduced into the system. Each resulting mixture was then vigorously stirred overnight at room temperature and left to stand for 48 h, allowing the mixtures to separate into two distinct phases.

#### 2.1.2. Preparation of water-in-water (W/W) LC emulsion

To study the LC properties of CNC in W/W emulsions, the separated isotropic and cholesteric phases of 5 % wt. CNC suspensions were incorporated into KC (10 % wt.) or CS (10 % wt.). The resulting phases containing the CNC/biopolymers, were collected and stored for further analysis. Briefly, the CS-in-KC W/W emulsions were prepared by mixing or sonicating each polymer individually with CNC using a vortex at a speed of 2000 rpm a speed of 2000 rpm a speed of 2000 rpm for 30 s along with 2 min of sonication. When CNC suspensions were added to the emulsions, they separated into an upper isotropic phase and a lower cholesteric phase during storage. The emulsion behavior was monitored after 7 days of storage at room temperature. The emulsions were then prepared in four different combinations based on the isotropic and cholesteric CNC/ polymer suspensions. Isotropic CNC/CS in cholesteric CNC/KC, cholesteric CNC/CS in isotropic CNC/KC, cholesteric CNC/CS in cholesteric CNC/KC, and isotropic CNC/CS in isotropic CNC/KC. To evaluate the effect of a reduced concentration of CNC (1% wt.) on the physical stability of W/W emulsions, we also utilized CS-to-KC volume ratios of 1:1 (E-1), 1:3 (E-3), 1:5 (E-5), and 1:7 (E-7). The emulsions were then analyzed for their physical properties, including the separation of phases, stability, and optical properties In all CNC/CS samples, curcumin was incorporated as a hydrophobic pharmaceutical compound.

#### 2.1.3. Calculation of the turbidity

The optical turbidity of the upper and lower phases of the CNC suspensions and emulsions was measured at 600 nm using the equation presented by Bai [28] in their study on the formulation and stabilization of water-in-water emulsions.

### 2.2. Microstructure microscopy

The microstructure of the prepared emulsion was studied using an Olympus BX53 optical microscope, equipped with a 10x or 20x air objective, and operated in fluorescence, phase contrast, and polarized modes. A small droplet of the aqueous emulsion was placed on a microscope slide and sealed with a glass coverslip to prevent evaporation and maintain the sample's integrity. To visualize the CS phase in the emulsion, we first added a fluorescent dye, CFW, to a suspension of CNCs in CS. This was done by stirring 10 µL of the dye into 5 mL of the CNC suspension, resulting in a uniform mixture of dye-labeled CNCs and CS. The excitation and emission spectra for CFW-labeled CNC and FITC-KC were measured as 365/435 nm and 493/517 nm, respectively.

Confocal laser scanning microscopy was performed with a Leica TCS SP5 microscope with Leica Application Suite AF using PMT detectors and an HCX PL APO CS 40.0 × 1.25 OIL UV objective with 405 nm laser for CNC-CFW excitation and 488 nm laser for FITC-KC excitation. A 488 nm argon laser was used as the light source combined with a 20 × or 40 × oil immersion objective in the absence of fluorescent labeling for polarized imaging [36]. The optical birefringence was collected in the transmission mode using a polarized laser and a linear polarized filter oriented perpendicular to the polarization plane of the incident light. The focused laser scanned the sample at a fixed depth and created a horizontal optical slice.

### 2.3. Printing process

In the development of each formulation, a 100-gram mixture of the drug and other components was prepared. To ensure proper particle size for printing, the powders were pre-sieved through a 180-µm mesh before being printed. The powder mixtures were then transferred to a desktop laser-powder bed fusion (L-PBF) printer (Sintratec Kit, AG, Brugg, Switzerland) for manufacturing oral dosage forms. The printer's platform reservoir (150 mm × 150 mm × 150 mm) was moved a sled to a building platform (also 150 mm × 150 mm × 150 mm), creating a uniform and level layer of powder. The printing surface temperatures ranged from 50 °C. A 2.3-watt blue diode laser (445 nm) was activated to bond the powder on the building platform according to a specific pattern based on the.stl file. As layers were added, the reservoir platform moved up, the building platform moved down, and the sled distributed a thin layer of powder. This process continued until the object was completed. The printlets were removed from the powder bed and excess powder was brushed off. Ten printlets were produced for each formulation.

## 2.4. Characterization of the printlets

### 2.4.1. Fourier-transform infrared spectroscopy (FTIR)

The transmission infrared spectra of 3D printlets and their precursors were identified with an FTIR spectrometer (Jasco FT/IR6200, Tokyo, Japan). The solid samples needed for the FTIR assay were obtained in the pellet form by blending approximately 10 mg of each sample with 100 mg of dry potassium bromide (KBr). Next, the samples were transferred to pellets to scan the spectral area at the wavenumber ranges of 400 and 4000 cm⁻¹, in which 50 scans were recorded with 1 cm⁻¹ resolution.

### 2.4.2. X-ray powder diffraction

In this study, discs measuring 23 mm in diameter and 1 mm in height were created from drug-excipient mixtures for analysis. Both pure paracetamol and the mixtures were examined. The X-ray powder diffraction patterns were obtained through the utilization of a Rigaku MiniFlex 600 instrument (Rigaku, USA), which features a Cu Kα X-ray source (λ = 1.5418 Å). The applied intensity and voltage were 15 mA and 40 kV, respectively. The data acquisition process involved an angular range of 3° to 60° (2θ), with a 0.02° step size and a scanning
speed of 5° min⁻¹.

### 2.4.3. Thermal analysis

The Differential Scanning Calorimetry (DSC) technique was employed for analyzing the powders and drug-loaded printlets. The DSC experiments were conducted using a Q2000 DSC device (TA Instruments, Waters, LLC, USA) with a heating rate of 10 °C/min. The instrument's calibration was done using indium (melting point 156.6 °C, enthalpy of fusion 28.71 J g⁻¹) as per the manufacturer's instructions. Nitrogen, with a flow rate of 50 mL min⁻¹, served as the purge gas for all experiments. The TA Advantage software for Q series (version 2.8.394) was used to collect data, which were later analyzed using TA Instruments Universal Analysis 2000 software. The reported melting temperatures are extrapolated onset values unless otherwise mentioned. The study utilized TA aluminum pans and lids (Tzero) with an average sample mass of 8–10 mg.

### 2.4.4. Determination of printlet morphology

In the process of analyzing and documenting the printlets, a digital caliper was employed to measure their diameter and thickness with precision. Simultaneously, a Sony α6300 camera was utilized to take photographs, providing visual documentation of these printed items.

### 2.4.5. Scanning electron microscopy (SEM)

To obtain comprehensive information about the printlets, surface and cross-section images were captured using a SEM model JSM-840A, manufactured by JEOL GmbH in Germany. Before conducting the SEM analysis, all samples were coated with a thin layer of carbon (approximately 30–40 nm) to improve their conductivity and enhance the quality of the images.

### 2.4.6. In-vitro drug release

The in vitro investigation focused on the release of curcumin from a 3D printlet, employing a modified diffusion approach as described by Rejinold [37]. To carry out the study, 3 mL of various 3D printlets with a concentration of 0.5 % w/v (F1-F5) were filled into cellulose dialysis bags featuring a molecular weight cutoff of 14 kDa. The sealed dialysis bags were then placed in vials containing 10 mL of phosphate buffer saline (PBS, pH 7.4) as the release medium. The vials were rotated at 100 rpm in a water bath (OLS Aqua Pro, Grant Instruments, Cambridgeshire, UK) maintained at 37 °C ± (0.5 °C).

Throughout the experiment, 1 mL aliquots of the release medium were withdrawn at specific time intervals (0.25, 0.5, 1, 2, 4, 6, 8, and 24 h) and replaced with equal volumes of fresh release medium. The cumulative percentage of released BH was determined by measuring its UV absorbance at 262 nm using a SynergyTM HTX microplate reader (BioTek, Winooski, VT). A standard curve of curcumin standards in PBS (pH 7.4) demonstrated a linear range of 0.1–50 µg mL⁻¹ with a high correlation coefficient (R²) of 0.99.

The mechanism of BH release from all formulations was examined by applying the zero-order, first-order, and Higuchi-diffusion equations [37–39]. The linear regression equation utilized for zero-order kinetics is represented as:

$$ Ct = Co - kt \quad (1) $$

In this equation, Ct denotes the amount of BH released at time t, Co refers to the initial concentration of BH, and k represents the zero-order release constant. By fitting the obtained data to these equations, the release kinetics of BH from the various formulations can be better understood and compared.

First-order kinetics were determined according to the equation:

$$ \ln C_t = \ln C_0 - kt \quad (2) $$

Drug release following the Higuchi model was determined using the equation:

$$ Q = kt^{0.5} \quad (3) $$

where Q represents the fraction of drug released in time t, and k is the Higuchi release rate constant.

### 2.5. Statistical analysis

All instrumental experiments were carried out as triplicate determinations and the mean and standard deviation of the data were reported. Analysis of variance (ANOVA) was utilized for the determination of the main effects of the examined independent factors and their interactions on the instrumental and sensory data. Duncan's multiple range test was applied to separate means of data when significant differences (P < 0.05) were observed.

## 3. Results and discussion

### 3.1. Unconventional phases of cholesteric LC: immiscible assembly and behavior

Through a controlled hydrolysis process using sulfuric acid, wood fibers were transformed into CNC of varying sizes [40]. These nano-particles exhibited rod-like shapes, measuring approximately 100–400 nm in length and 8–12 nm in diameter (Figure S1, Supporting Information). The particles had highly electronegative charges, with a ζ-potential of –49.3 mV. The phase separation of CNCs was evaluated in the presence of hydrophilic, coiled KC and CS (Fig. 1, and Figure S2 in Supporting Information). As anticipated, there was no birefringence detected in the low particle concentration of CNC suspension (i.e., 2.0 and 4.0 % wt.), indicating an isotropic system (as further supported by Figure S3, Supporting Information).

A phase separation took place upon increasing the CNC concentration to 5.0 % wt, which resulted in the formation of a lower cholesteric phase and an upper isotropic phase. In contrast, the presence of either CS or KC in the CNC suspension caused a transition to a biphasic system, as clearly illustrated in Fig. 1, Columns i and ii. In the context of cholesteric phase behavior studies, the volume fraction of CNCs notably decreased from the initial value of 0.618 to 0.576 and 0.224 for CNC/CS (including curcumin) and CNC/KC systems, respectively (Figure S2, Supporting Information). This reduction in volume fraction could signify an increased relative enrichment of CNC particles within the anisotropic phase [28]. The qualitative assessment, conducted using turbidimetry, demonstrated a notable improvement from an initial value of 0.478 to 0.592 for the CNC/CS system and 0.616 for the CNC/KC system (Section S2, Supporting Information). The phenomenon observed in the study involved the widening of the biphasic area and the conspicuous enrichment of CNCs within the cholesteric phase. This intriguing outcome can be attributed to the mutual exclusion impact that arises due to the interplay between the rod-like nanoparticles, which are the CNCs, and the non-adsorbing biopolymer coils [41]. This interaction between the two components may lead to the observed alterations in the phase behavior and the consequent enrichment of CNCs in the cholesteric phase [28].

Flory's calculations provide a profound insight into the behavior of anisotropic LC phases and their selectivity towards foreign components [42]. These phases, as they approach the selectivity of a pure crystal, exhibit a remarkable ability to reject foreign components. Conversely, the isotropic counterpart is more accommodating, allowing the incorporation of these foreign components. In the context of the biphasic suspension containing CNC, CS, and KC, the addition of either CS or KC can lead to an interesting phenomenon. These foreign components replaced CNCs in the isotropic phase, thereby increasing the concentration of rod-like particles in the anisotropic phase. This substitution process results in an inhomogeneous suspension, with a higher concentration of CNCs in the cholesteric phase and a lower concentration in
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Phase separation of CNC/biopolymer suspensions with a cholesteric organization. (Columns *i* and *ii*) Photographs of the CNC (5.0 % wt.) and the binary suspension after incorporation of either CS or KC. (Column *iii*) POM images of the cholesteric phase for CNC alone, CNC/CS (including curcumin), and CNC/KC suspensions, presenting periodic fingerprint patterns. (Column *iv*) Histogram and distribution of the helical pitch of CNC alone, CNC/CS, and CNC/KC suspensions.</figcaption></figure>

the isotropic phase [28]. This inhomogeneity is a direct consequence of the interplay between the selectivity of the anisotropic LC phase and the incorporation behavior of the isotropic phase, as described by Flory's calculations.

The inhomogeneous suspension exhibits a distinct partitioning pattern between the upper and lower phases. The upper region is characterized by a higher concentration of the non-adsorbing biopolymer coils, while the lower region contains a relatively lower concentration of CNCs. This uneven distribution is a direct consequence of the aforementioned substitution process involving CS or KC. Comparing this scenario with a biphasic system derived from a CNC suspension reveals a significant difference [28]. In the latter case, the phase separation occurs without the presence of CS or KC. However, when these foreign components are introduced and dispersed into the fully anisotropic phase, they do not seem to have any noticeable impact on the phase separation process (data not shown). This observation implies that the non-adsorbing biopolymeric coils can tolerate and coexist with the cholesteric LC phase, maintaining their structural integrity and properties.

The polarized optical microscopy (POM) images of the cholesteric LC before and after the addition of either CS or KC are shown in Fig. 1, Column *iii*. A continuous long-range fingerprint pattern with a minimal number of morphological irregularities was found for the pure CNC cholesteric phase. A similar result was reported for the formation of continuous fingerprint texture within CNCs in a cholesteric LC system [28]. This pattern is a typical representation of the uniform helical organization that extends across extensive distances within the material.

Upon incorporating CS and especially KC into the CNC cholesteric phase, the helical pitch significantly decreased from an initial value of 17.4 µm to 13.8 or 8.2 µm regarding CS and KC, respectively, as shown in the histograms of Fig. 1. Column iv. Consequently, the fingerprint texture transformed, featuring a convoluted pathway and an increased presence of dislocations and disclinations. The dislocations observed in the cholesteric phase can be linked to the introduction of foreign biopolymers, which introduce internal constraints. These constraints may not be easily accommodated by the continuous reorientation of the LC director through bending, twisting, or splaying. Consequently, the presence of these defects in the LC phase leads to a distribution of lower free energy, primarily concentrated at the core of these imperfections. In summary, the average gap between the adjacent CNC nanorods in the cholesteric phase varies from 25 to 51 nm depending on concentration [28,43]. CS and KC hydrodynamic radii were detected to be 9.1 and 19.1 nm, respectively, which are smaller than the CNC separation distance (Figure S4). Due to their size, shape, and high concentration, biopolymers can be inserted between CNCs in the cholesteric structure. Excess biopolymers may cause distortions in the cholesteric LC and enrich within topological defects [28].

## 3.2. Cholesteric assembly in immiscible phases

Unlike conventional oil-in-water (O/W) emulsions, there was the occurrence of an inferior interfacial tension at the W/W interface within the all-aqueous CS/KC bicontinuous emulsion. This phenomenon is facilitated by Rayleigh-Plateau instability, which generates liquid jets
instead of droplets (data not shown). In this context, the introduction of non-adsorbing biopolymers like CS and KC as depletants to the CNC suspension can substantially affect the isotropic-cholesteric phase transition, as mentioned in the study by Bai et al [28]. These biopolymers play a crucial role in altering the properties of the CNC suspension, leading to changes in the phase behavior and overall structure [28]. In the cholesteric phase, the CS/KC interfacial region displayed a noticeable, bright, and birefringent appearance in the magnified POM micrographs (Fig. 2a). The isotropic phase showcased visual extinction, indicating the separation of CNC/biopolymer systems. Blending an isotropic CNC/CS complex coacervates with another isotropic CNC/KC suspension resulting in a demixed profile between the two phases (Figure S7). Please note that in the CNC/CS hydrophobic curcumin as a drug model existed. The lower CNC/KC phase transformed into an anisotropic state upon reaching equilibrium, suggesting a slight migration of CNC from the CS phase to the KC phase. Despite the visibly distinguishable W/W interface in the CNC/biopolymer suspensions at various scales, it represents a molecular-level interphase with a thickness surpassing conventional oil/water interface [44]. A gradient in

biopolymer concentration is anticipated at the nanoscale level, with the overall biopolymer concentration at the interfacial region showing a local minimum due to repulsion between individual polymers. The transition in the polymer contents, starting from the bulk value in the CS phase and progressing to the bulk value in the KC phase, occurred gradually over tens of nanometers [45], similar to the distance between individual CNCs, enabling short-range repulsive interactions for CNCs at the W/W interfacial zone.

Despite being cholesteric or isotropic in structure, when the CNC/KC suspension was mixed with the CNC/CS (containing curcumin) at a 1:3 vol ratio, a binary aqueous system was formed. As Fig. 2a depicted, this system possessed a distinct phase boundary at the W/W interface, forming a transparent aqueous emulsion. This all-aqueous, bicontinuous emulsion, comprised KC and CS with a level of 10% wt. Both phases also contained identical concentrations of CNC (5 % wt.), which formed cholesteric-cholesteric, isotropic-cholesteric, or cholesteric-isotropic binary combinations. The phase separation phenomenon could be attributed to the repulsive interactions between KC and CS molecules, which exhibit a limited mixing entropy (pertaining to the samples

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** A transition from macro to micro: Binary W/W emulsion with anisotropy. (a) Images of demixed CNC/CS (upper) and CNC/KC (lower) binary suspensions, showing a clear phase boundary and different LC combinations. (b) POM images of the demixed suspensions, focusing on the phase boundary, revealing birefringent textures in the cholesteric LC phase and optical extinction in the isotropic phase. (c) After vortex agitation or sonication, a homogeneous W/W emulsion is obtained. (d) Low- and high-magnification optical images of the prepared emulsions show polydisperse droplets with slightly spherical shapes. The inset displays the emulsion droplet size distribution. (e) After 2 days of equilibrium, the emulsions separate into an upper isotropic phase and a lower anisotropic phase. (f) The calculated ratio of the anisotropic phase for emulsions *i-iii* before and after emulsification is shown. Turbidity measurements of the separated emulsion *i* (cholesteric-to-cholesteric) and CNC suspension, upper and lower phases, indicate that the droplets are enriched in the lower phase. The values in (f) were calculated from the turbidity ratio of the lower and upper phases.</figcaption></figure>
subjected solely to vortex mixing) [28]. This is compensated by the positive mixing enthalpy in water, promoting segregation beyond certain concentrations [46]. Detailed POM images, when magnified, showcased a clear boundary with bright, birefringent textures in the cholesteric phase and optical extinction in the isotropic counterpart (Fig. 2b), confirming the demixing of the CNC/biopolymer suspensions. Contrastingly, when an isotropic CNC/CS suspension was layered over another CNC/KC isotropic suspension, a demixed profile emerged between the two isotropic counterparts. Upon reaching equilibrium, the bottom CNC/KC phase transformed into an anisotropic state, which can indicate the partial migration of CNC from the CS phase to the KC phase (Figure S5). Although the W/W interface in the CNC/biopolymer suspensions may seem well-defined visually at both macroscopic and microscopic scales, it should be noted that this interface is, in fact, an interphase at the molecular level. This interface is significantly thicker compared to typical O/W interfaces [44]. At the interfacial region, the overall biopolymer concentration exhibited a local minimum due to the repulsion between each polymer molecule, which hindered interpenetration.

Investigating the optical microscope attributes of the aqueous emulsion featuring CNC within binary CS/KC polymer compositions can offer valuable perspectives on the colloidal self-assembly process and the intricate internal structure. To generate W/W emulsions, we combined 10 % CS and 10 % KC within the aqueous CNC/biopolymer mixtures using vortexing or sonicating, achieving a consistent and well-mixed W/W emulsion. The prepared systems displayed a visually homogeneous aspect, as illustrated in Fig. 2c. To commence, the CNC/CS (containing curcumin) phase functioned as the dispersed phase in the continuous CNC/KC phase was verified. This resulted in the creation of spherical CNC/CS droplets (Fig. 2d, Top). Subsequently, the size distribution and colloidal stability of the emulsion droplets were evaluated. To accomplish this, a W/W emulsion was prepared, which allowed it to remain undisturbed in a glass cuvette under ambient circumstances. Primarily, this system offered a polydisperse droplet of CNC/CS with a slightly uniform size (Fig. 2d, Bottom). Upon closer examination using optical magnification, some spherical droplets were observed with average diameters varying from 8 to 16 µm. Furthermore, a distinct fingerprint pattern was also found in the vicinity of the emulsion droplets, which is a sign of the CNC self-assembly to the cholesteric arrangement (red arrows in Fig. 2d, Bottom).

Three as-prepared distinct emulsions using different LC phase combinations, including cholesteric (CS)-to-cholesteric (KC), isotropic (CS)-to-cholesteric (KC), and cholesteric (CS)-to-isotropic (KC) are shown in Fig. 2e (from left to right), which were named as emulsion 'i', 'ii', and 'iii'. All of the emulsions underwent phase separation, resulting in the formation of two coexisting phases. The obtained W/W emulsions underwent phase separation over a period of 48 h, resulting in the formation of two distinct phases: a clear, isotropic upper phase and a turbid, anisotropic lower phase (Fig. 2e). Notably, the droplet size and distribution of the emulsions after 7 days remained slightly unchanged compared to the freshly prepared samples, indicating a stable W/W system (Figure S6). It should be noted that the stability behavior of the created W/W emulsion contrasted with the usual Pickering stabilization effect, where CNC would specifically assemble at the interface between CS and KC. To quantify the phase separation, the volume fraction of the anisotropic phase ($\phi$) was calculated before and after complete phase separation (Fig. 2f, Left). Before emulsification, emulsions i and ii showed an $\phi$ of about 1.0 and 0.8, whose values decreased to 0.6 and 0.4 after emulsification, while $\phi$ value of emulsion iii from an initial value of 0.35 increased to 0.42. These results suggest that the presence of the two hydrophilic biopolymers induced a re-equilibrium in the CNC cholesteric assembly. The droplet distributions in the two phases were characterized by measuring turbidity through regular optical image counting (Fig. 2f, Right). The results showed that all of the emulsions and initial CNC suspensions exhibited larger turbidity in the bottom phase, whereas the upper phase was translucent (Figure S7). The turbidity ratio of the

bottom phase to the upper phase was found to be 1.24 and 1.86 for emulsion i and the initial CNC suspension, respectively (Fig. 2g). This disparity suggests that the droplets in the emulsion i bottom phase are considerably enriched, as the turbidity of this phase is greater than that of the binary CNC suspension.

### 3.3. Bicontinuous structured LC emulsion: morphological characterization

We generated a homogeneous W/W emulsion by vortexing or sonifying cholesteric CNC/CS suspension (containing curcumin) and cholesteric CNC/KC counterpart at different volume ratios of 1:1 (E-1), 1:3 (E-3), 1:5 (E-5), and 1:7 (E-7) (Fig. 3, column i), which contained CS and KC with a level of 10 % wt. and identical concentrations of CNC (2 % wt.), which we aim to reduce the amount of CNC to monitor its effect on W/W emulsion stability. To visualize the emulsion's structure, a calcofluor-white (CFW) stain was used to label the CNC surface and FITC molecules to tag the KC biopolymer. FITC-KC exhibits excitation and emission peaks at 490 nm and 520 nm, while CFW has peaks at 380 nm and 475 nm. Consequently, both CNC and KC can be labeled with distinct fluorophores, serving as separate fluorescent markers. These markers exhibit minimal cross-influence on each other's fluorescence properties, allowing for independent observation and analysis of their respective characteristics. The depicted image in Fig. 3 showcases a set of fluorescent microscopy captures of a freshly prepared emulsion (Fig. 3, column ii) featuring a 1:100 ratio of two distinct fluorescent markers. Post-homogenization, the resulting all-aqueous emulsion displays a bicontinuous structure, characterized by an extensive network of interpenetrating liquid domains with a size of several hundred micrometers. The bicontinuous emulsion containing fluorescent markers demonstrated a disparate fluorescence distribution upon excitation. In this particular instance, one of the interconnected regions within the emulsion displays a higher fluorescence intensity than the other (Fig. 3, column iii). With the excitation wavelength set to 493 nm, the fluorescent emission of FITC-KC takes precedence over that of CFW-labeled CNC, with the former becoming more prominent. Under the specified conditions, the fluorescence examination successfully distinguished the CS domain and the immiscible binary polymer mixture, with the CNC domain appearing dark (Fig. 3, column iv). It is crucial to mention that a higher relative fluorescence intensity corresponds to an increased concentration of CFW-labeled CNC and FITC-KC. This observation suggests that the intricate CS domain contains a greater number of CNC compared to the KC domain, signifying an uneven distribution of CNC within the binary polymer mixture (Fig. 3, column v). The distribution of CNC within the multidomain structure of this bicontinuous system can be further evaluated by analyzing the fluorescence intensity (Figure S8). The results obtained exhibit high CFW fluorescence in the CS region and a noticeable absence of fluorescence in the KC region for FITC-KC. This confirms the encapsulation of CNC within the CS domain of the bicontinuous emulsion. In the present scenario, the varying affinity of CNC nanoparticles for the CS and KC domains in the bicontinuous emulsion can be attributed to the equilibrium between osmotic potential and chemical potential within the two biopolymer phases. The CS domain, characterized by a lower polymer potential compared to its KC counterpart, results in the enhanced ingress of CNCs into the CS region of the binary polymer blend, leading to the interphase retention of nanoparticles.

The POM imaging was utilized to observe the self-assembly of CNC between the dispersed CS phase and continuous KC phase. Initially, the colloidal dispersions of E-1 and E-3 displayed an anisotropic pattern due to the separation of polymeric colloidal dispersions (Fig. 4, column i). However, the formation of spherical-like and elongated birefringent tactoid near shape with fingerprint patterns was detected in the E-5 and E-7, as the cholesteric CNC/CS droplets transitioned into an isotropic state. The emergence of these tactoids in an isotropic phase can be explained by the increased particle concentration of KC and the aggregation of particles into a microdroplet domain with liquid-crystalline
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Photographs of (i) fresh and (ii) homogeneous W/W emulsion by vortexing/sonicating cholesteric CNC/CS suspension and cholesteric CNC/KC counterpart at different volume ratios. (iii-v) Fluorescence microscopy images of the homogeneous bicontinuous LC emulsion that focus on the CFW-labelled CNC, FITC-KC, and their composite representation, respectively.</figcaption></figure>

ordering. The transfer of CNC from the cholesteric CNC/CS phase to the cholesteric CNC/KC phase through diffusion might disrupt the balance in the system and lead to the reassembly of CNC in W/W emulsions. In conclusion, the POM results indicate that osmotic pressure plays a role in driving the permeable self-assembly of CNC between the dispersed CS phase and the continuous KC phase. As observed 6 h after emulsification, the mixed emulsions showed a decrease in droplet size and distribution (Fig. 4, column ii).

To gain more insight into the development of emulsion morphology, we initiated the fluorescent investigation by systematically adjusting the initial volume ratio. Upon emulsification, the CS dyed with RBITC exhibited fluorescence, while the KC droplets displayed reduced fluorescence. As the emulsion's morphology is intricately linked to the polymer composition in aqueous solutions, it is possible to transform the emulsion's morphology from a bicontinuous structure to aspherical droplets by altering the ratio of CS to KC or the initial polymer concentration. In Fig. 4, column iii, the resulting E-1 (and some local area of E-3) emulsions exhibited tortuous bicontinuous domains, which transformed into droplet morphology regarding E-5 and E-7, featuring aspherical RBITC-CS droplets within the KC continuous phase (Fig. 4, column iii). CS is a positively charged polysaccharide, while CNC is negatively charged, leading to the formation of a robust composite gel of CNC/CS in the form of polydisperse droplets with slightly spherical

shapes within the continuous phase. To study the hierarchical self-assembly of CNC particles in a complex emulsion, we employed non-invasive polarized laser scanning confocal microscopy (polarized-LSCM) in polarization mode across the sample (Fig. 4, column iv), investigating the cholesteric self-assembly of CNC particles. The high-resolution polarized LCM imaging revealed a microscale fingerprint pattern within the interconnected liquid network, confirming the hierarchical cholesteric self-assembly of CNC particles. The optical results offer a unique window into the internal structure and colloidal self-assembly of the all-aqueous bicontinuous LC emulsion. The analysis reveals the presence of a three-dimensional, percolating network of cholesteric LC in the isotropic phase, which is formed through the intraphase trapping and self-assembly of CNC within binary polymer domains.

### 3.4. Formation of encapsulated all-aqueous emulsions

To create microcapsules including curcumin from oppositely charged polyelectrolytes, it is highlighted that complexation may occur between CNC and CS [30–33]. To gain a clearer understanding of the composition of these phases within the microcapsules and to pinpoint the location of the nanoparticles, we conducted experiments using fluororescently labeled CS, KC, and CNC (Fig. 5 a-c). The results indicate that
<figure><img src="image_7.png" /><figcaption>Fig. 4. (i) POM imaging of homogeneous W/W emulsion, (ii) droplet size and distribution. (iii) Fluorescence images of different W/W emulsions. (iv) Non-invasive polarized-laser scanning confocal microscopy (polarized-LSCM) in polarization mode.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 5. Components labeled with fluorescent markers are incorporated during the creation of emulsion microcapsules. (a) Rhodamine-tagged CS. (b) False-colored tetramethylrhodamine isothiocyanate (TRITC) mixed isomers in the core CNC. (c) False-colored rhodamine-tagged KC. Insets depict the corresponding bright field images. (d) Schematic representation of the all-aqueous emulsion formation, illustrating the initial positioning of the CNC and polyelectrolytes within the systems. Photomicrographs from CLSM (e) and SEM (f) reveal the CS- and KC-rich phases that comprise the dispersed and continuous phases in the all-aqueous emulsion. All scale bars measure 30 µm.</figcaption></figure>
the internal droplet is predominantly composed of CS/CNC, whereas the continuous phase is rich in KC/CNC. Importantly, the nanoparticles are also present in the encapsulating membrane, suggesting that the interface of the inner droplet consists of CS along with nanoparticles. Notably, we observed internal droplets present within the CS/CNC structure, where the outer layer of these droplets was also covered by a shell, while the internal droplets displayed a spherical shape, resembling droplets found in an immiscible liquid system (Fig. 5 d-f). Although the two phases within the irregularly shaped shell seem to correlate with those used to create the W/W emulsion system, their specific characteristics remain unclear in the photomicrographs. The SEM analysis (data not shown) shows that the thickness of the shell is approximately 3–6 µm, a measurement similar to those previously reported for

microcapsules produced through the interfacial complexation of alumina-coated silica [33].

### 3.5. Characterization of the printlets

#### 3.5.1. SEM images of freeze-dried microspheres

We utilized a W/W LC emulsion encapsulating curcumin for our 3D/4D printing applications. The primary objective of this study was to leverage L-PBF technology to fabricate 3D/4D porous printlets. In the L-PBF process, layers of powder-based materials are deposited, melted, and solidified using a laser, forming a solid 3D layers. This process is repeated with consecutive layers being joined to form fully solid 3D components. To construct the desired 3D/4D printed structures through

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** (row, i) SEM images of the freeze-dried LC-based powders. (row ii) Photomicrographs of 3D/4D printlets. (row iii) FE-SEM images of the surface of 3D/4D printlets. (rows iv and v) Micro-CT to investigate the 3D pore structure and internal dynamics of the 3D printlets. The samples with different cholesteric CNC/CS and cholesteric CNC/KC volume ratios of 1:1, 1:3, 1:5, and 1:7 were labeled as E-1, E-3, E-5, and E-7, respectively.</figcaption></figure>
L-PBF, the LC-based emulsions (E-1, E-3, E-5, and E-7), exhibiting bicontinuous architecture, were spray-dried and converted into microsphere powders before printing. The SEM images of the freeze-dried powders are displayed in Fig. 6, row i, showcasing variations in granule size, size distribution, and shape. All powder types exhibited a large, rounded morphology. The E-1 sample demonstrated a more diverse size distribution and a ball-shaped appearance, with a size distribution characterized by d₁₀ of 0.5 µm, d₅₀ of 34 µm, and d₉₀ of 42 µm. Some powder samples adhered together, particularly noticeable in E-3 and E-5. E-3 powders consisted of a mixture of square and round granules, while the E-5 sample had predominantly disk-shaped structures with a uniform blend. The E-7 sample appeared round and contained small square-shaped components. Additionally, numerous spots and pores were observed on the exterior of the powders. The granule size variation detected through SEM was consistent with the measured granule size distribution, which had a d₁₀ of 0.3 µm, d₅₀ of 12 µm, and d₉₀ of 16 µm.

### 3.5.2. Printing process

The primary objective of this work was to employ laser-powder bed fusion (L-PBF) 3D printing to manufacture printlets (3D printed tablets) to develop customizable drug release profiles. To manufacture the printlets, four unique powder compositions were developed, each consisting of 90 % LC-derived powders and 10 % curcumin. The L-PBF technique was employed to fabricate these printlets since it involves the fusion of powder particles through thermal binding, where a laser beam traces the 3D design by drawing on the powder bed. To attain optimal print quality, it is crucial to find the ideal equilibrium between the printer's internal temperature and the laser scanning speed [47]. By reducing the laser scanning speed, a prolonged interaction period between the powder particles and the laser beam ensues, resulting in increased energy transmission. This can lead to the production of denser and stronger objects. In contrast, increasing the laser scanning speed diminishes the energy transmitted, causing the formation of weaker and more porous printlets.

L-PBF technology offers two significant advantages for manufacturing intricate drug products. First, it enables the fabrication of highly accurate structures due to the precision of the laser utilized in the printer, which is contingent upon the laser's capabilities and the size of the powder particles. Second, it obviates the need for additional support structures during the printing process, as the unsintered powder serves as its own support. This maintains the object's integrity and averts potential shape irregularities or dose inaccuracies related to support removal in other printing technologies. The E-1 printlet was brittle and deformed during the tensile strength test (Fig. 6, row ii), which broke into small pieces during the analysis, preventing the determination of their breaking force value. This resulted in an undetectable breaking force value. In contrast, the E-3 and E-5 printlets were strong, demonstrating similar breaking forces of approximately 319 N. The printlet of

<table><caption>**Table 1**

Physicochemical characteristics of 3D printed printlets.</caption><thead><tr><th>Sample</th><th>Weight (mg)</th><th>Moisture content (%)</th><th>Drug content (%)</th><th>Porosity (%)</th><th>Breaking force (N)</th></tr></thead><tbody><tr><td>E-1</td><td>328.4 ± 1.8<sup>d</sup></td><td>2.12 ± 0.02<sup>a</sup></td><td>80.4 ± 1.6<sup>a</sup></td><td>12.3 ± 0.1<sup>a</sup></td><td>nd</td></tr><tr><td>E-3</td><td>319.6 ± 1.8<sup>c</sup></td><td>2.10 ± 0.04<sup>a</sup></td><td>90.4 ± 1.4<sup>b</sup></td><td>18.4 ± 0.7<sup>b</sup></td><td>318.7 ± 6.9<sup>a</sup></td></tr><tr><td>E-5</td><td>310.4 ± 1.6<sup>b</sup></td><td>3.06 ± 0.04<sup>b</sup></td><td>96.3 ± 1.4<sup>c</sup></td><td>33.1 ± 0.5<sup>c</sup></td><td>319.3 ± 7.3<sup>a</sup></td></tr><tr><td>E-7</td><td>294.6 ± 1.7<sup>a</sup></td><td>3.43 ± 0.05<sup>c</sup></td><td>99.3 ± 1.7<sup>d</sup></td><td>47.1 ± 0.7<sup>d</sup></td><td>379.3 ± 6.8<sup>b</sup></td></tr></tbody></table>

E-7 was more robust with the highest breaking force values of around 379 N (Table 1).

The microstructure of the printlet is a crucial factor in its functionality as a drug delivery platform since it significantly impacts the rate of water permeation and drug release. The pore size and shape of the printlets matrix play a vital role in these processes, as they influence the transport of molecules and the release of therapeutic agents. In this context, FE-SEM was employed to examine the microstructure of the freeze-dried printlets (Fig. 6, row iii). The SEM micrographs revealed that all hydrogel scaffolds exhibited a porous structure. The microstructure of the E-1 presented a low-level porous structure with low sizes. Whereas, E-3 yielded a coarser and rougher surface morphology, accompanied by more compact porous structures. Also, the pores of E-3 are largely closed and show no interconnections. Notably, the micrographs showed that the E-5 and E-7 exhibited a high-level of porous structure than those E-3. These findings suggest that a highly porous structure can be formed in a bicontinuous emulsion sample with a percolating network of cholesteric LC in the isotropic phase. Moreover, these results indicate that highly porous structures can be tailored by the intraphase trapping and self-assembly of CNC within binary polymer domains.

Pore characteristics, such as porosity, pore size distribution, and pore geometry, are crucial factors to examine when studying the microstructure of 3D printlets. By employing micro-CT to investigate the 3D pore structure and internal dynamics of the 3D printlets, one can assess the distribution of porous structure by focusing on the 3D pore structure (Fig. 6, rows iv and v). The green color indicates larger pores based on their volume, and red represents smaller pores. Initially, the prominent red pore in E-1 consists of an elongated, dissolved shell fragment, surrounded by smaller pores. Regarding E-3, the red volume appeared, with relatively larger pores (green) at the sample's edges. Eventually, E-5 and E-7 showed a large shell fragment connected with the newly formed pore structure on the border, which in the case of E-7 resulted in a single, extensive pore structure (red).

### 3.5.3. FTIR

A thorough molecular investigation was conducted using FTIR analysis to study the interactions between various polymeric materials present in the 3D object (Fig. 7a). The results revealed common bands in the range of 3283–3350 cm⁻¹ for all 3D printlets, which are associated with OH stretching and bending frequencies in 3D printlets [48–50]. Moreover, some peaks were detected at 2877 and 2865 cm⁻¹ that correspond to the symmetric C-H₂ stretching [51]. According to the pure CS and KC spectra, signals at 1250, 930, and 840 cm⁻¹ signify the presence of significant bonds, such as ester sulfate group (S=O stretching vibration), 3,6-anyhydride galactose group (bending vibration), and D-galactose-4-sulfate (stretching vibration) [52]. Moreover, the wavenumbers 1646 cm⁻¹ and 1570 cm⁻¹ regarding the pure chitosan spectrum denote the stretching vibrations of –NH and –NH₂ groups, as stated by Guan et al [53]. Additional characteristic peaks include C-H stretching (2936 cm⁻¹), asymmetric COOH stretching (1637 cm⁻¹), CH₂ symmetrical bending (1426 cm⁻¹), and notable bands at 1075 cm⁻¹ and 962 cm⁻¹, indicative of cellulose C-O-C bridges and ether C-O-C functionalities, respectively, signifying the attendance of cellulose I [54]. The FTIR spectrum of curcumin shows prominent peaks attributed to the stretching vibration of the phenol-OH group at 3505 cm⁻¹, conjugated C=O stretching at 1628 cm⁻¹, C=C stretching at 1510 cm⁻¹, and the olefins C-H bending vibration at 1429 cm⁻¹ [55].

Alternatively, the L-PBF has facilitated etherification between the –OH groups, predominantly in the 3D printlets variants. The FTIR spectra of the 3D printlet including curcumin displayed noticeable differences compared to precursor compounds. The peak at 1383 cm⁻¹ is attributed to C-H bonds in methyl groups (associated with acetamide moieties), confirming that CS is a part of the structure. The peak at 1526 cm⁻¹ was also related to –NH₃⁺ groups in CS. The peak at 1638 cm⁻¹ is assigned to the carboxylate anion in KC. The FTIR spectrum of the 3D

<footnote>*nd: not detected.</footnote>

<footnote>The means in each column with various letters (a–d) are significantly different (p < 0.05) according to Duncan's test.</footnote>
<figure><img src="image_10.png" /><figcaption>Fig. 7. (a): FTIR spectra, (b): XRD patterns (c) TGA analysis, and (d) DSC analysis of 3D/4D printed printlet and its components.</figcaption></figure>

printlet showed characteristic peaks of curcumin. The peak at 902 cm⁻¹ in the curcumin spectrum shifts to 897 cm⁻¹ in the 3D printlet spectrum, and its intensity decreases. Similarly, the peak at 823 cm⁻¹ shifts to 803 cm⁻¹ in the FTIR spectra. The absorption bands at 2918 cm⁻¹ and 1032 cm⁻¹ in the 3D printlet spectrum could be attributed to C-H and C=C stretching, respectively. The vibration of C-O-C and C-C bonds, detected at 1260 cm⁻¹ in all 3D printlets, provides indirect proof of physical crosslinking during the L-PBF process. The signal at 1639 cm⁻¹ in the 3D printlet results from the overlapping of C=O and C=C bonds in RC with -NH₂ groups, as well as amide III at 1321 cm⁻¹. Furthermore, the disappearance of the prominent -OH peak at 2283 cm⁻¹ in the 3D printlet suggests the formation of intermolecular hydrogen bonds between the oxygen in the C=O double bond of curcumin and the hydrogen in the -OH groups of CS, CNC or KC, due to their similar molecular structures. These observed changes support 3D printlet formation, indicating that curcumin is present in the samples.

### 3.5.4. X-ray diffraction (XRD)

Fig. 7b illustrates the XRD patterns of 3D printlets and their precursors. Notably, CS primarily exhibits an amorphous structure, whose XRD profile reveals two characteristic crystalline peaks at 2θ = 10.8° and 22.9°, which correspond to a partial crystalline structure and hydrated crystals originating from the α-chitin present in the pure CS sample. In the depicted Fig. 7b, the XRD pattern for pure CNC exhibits notable peaks at 2θ angles of 15.0°, 16.5°, 22.6°, and 34.6°. These peaks align with the Miller indices (110), (-110), (002), and (040), confirming the presence of cellulose I. The XRD pattern of the raw curcumin crystal exhibited characteristic peaks at 2θ of 8.98°, 12.26°, 14.62°, 17.33°, and 24.51°, indicating the presence of crystalline phases.

During the L-PBF printing process, the appearance of broad peaks at 2θ angles of 13.8° and 25.9° suggests the formation of semi-crystalline materials. This finding is consistent with the data obtained from DSC. The crystallinity patterns of curcumin are somewhat evident in the diffractograms of the 3D printlet. The characteristic peaks of curcumin
are indicative of a high crystalline structure. In the case of curcumin loaded into a 3D printlet, peaks at 16.4°, 12.8°, 26.5°, and 30.0° are not observed, likely due to the formation of an amorphous complex resulting from intermolecular interactions between biomolecules [56]. The intensity of the peak at 2θ = 20.1° in the 3D printlet becomes weaker and broader. Additionally, a new peak at 2θ = 6.9° indicates good compatibility between the two polymers.

### 3.5.5. TGA and DSC

The thermal degradation of the 3D printlet and its precursors was studied through thermogravimetric analysis, as shown in Fig. 7c. The pyrolysis process involved two stages for all biopolymers and 3D printlets. In the case of CS, the TGA curve indicated an initial weight loss at 320 °C, which could be attributed to the loss of absorbed water. As the temperature increased up to 370 °C, a weight loss of 40 % was observed, which signifies the degradation of the polymer backbone and the complete degradation of CS at 450 °C. The TGA curve for pure KC also showed a 10 % weight loss at 150 °C and approximately 30 % weight loss as the temperature increased from 250 °C to 310 °C, which can be attributed to the degradation of the carbohydrate structure. As the temperature rose from 310 °C to 450 °C, the complete degradation of the polymer occurred, possibly due to the scissions of the polymer backbone. In contrast, the TGA curve of 3D printlets revealed a 20 % weight loss at 360 °C, indicating high thermal stability. Furthermore, 50 % weight loss occurred between 360 °C and 450 °C, after which the 3D printlets network was fully degraded. However, the thermal degradation profile of the 3D printlet was much lower, indicating that the production of the 3D structure altered its properties.

DSC analysis was conducted on CS, KC, CNC, and curcumin, and formulated 3D printlet to study their thermal behavior (Fig. 7d). The thermogram of CS displayed a typical endothermic peak of about 95 °C attributed to the removal of adsorbed water, followed by a sharp endothermic peak at 230 °C associated with a restricted local depolymerization. A broad exothermic peak appeared at about 326 °C, which corresponds to the owing to cold-crystallization region, as mentioned in the reference. The DSC curve of KC exhibited an endothermic peak of approximately 100 °C, which can be attributed to the dehydration of the polymer. Additionally, a sharp endothermic peak was observed at 425 °C and was assigned to the restricted local degradation of the polymer network. In contrast, the DSC curve of CNC showed an endothermic transition temperature of 110 °C, which can be attributed to the water evaporation and featured a clear endothermic peak at about 340 °C. Interestingly, the first endothermic behavior of the non-printed sample was found to be 135 °C, which is higher than that of all the pure polymers. This increase in this peak can be credited to the formation of hydrogen bonding between the hydrophilic moieties of CS and KC and the OH group of the CNC chain [51]. The DSC curve of this sample also exhibited a lower exothermic peak at 230 °C and a broad endothermic peak at about 330 °C, which indicates the development of some kind of physical interactions non-printed sample. Alternatively, the DSC profile of the 3D printlet exhibits the same endothermic peak of pure curcumin at about 185, 230, and 325 °C, but with reduced intensity. The broad and narrow endothermic peak in the DSC curve indicates that curcumin was included well in the 3D printlet during the L-PBF process.

### 3.5.6. Thermoresponsivity of 3D printlets

Although pure CS and KC are not typically regarded as “perfect” thermoresponsive materials, recent studies have demonstrated that KC can exhibit thermoresponsive behavior under certain conditions. Specifically, KC has been shown to possess thermoresponsive characteristics, with studies reporting its use in developing thermo-responsive gels that are sensitive within a temperature range suitable for living cells (37–45 °C) and capable of reversible volume transitions in response to thermal stimuli [54]. The size of KC-based nanoparticles can be tailored by varying KC concentration while keeping surfactant concentration constant, primarily due to electrostatic interactions between the

oppositely charged surfactant and KC's sulfate groups. Additionally, blending KC with other biopolymers has been found to induce a sol–gel transition within the physiological temperature range (30–60 °C), facilitating the entrapment and release of therapeutic agents [40,55].

In the experimental setup, the optical examination revealed a discernible variation in cloud point among the reference, the upper layer, and the lower layer (Fig. 8). Turbidity measurements further validated these findings. The thermoresponsive nature of the synthesized 3D printlet and its precursors is primarily driven by the temperature-induced solubility change of the CS and KC. As the temperature rises above the cloud point (Tc), the CS and KC are switched from hydrophilic to hydrophobic, which leads to a conformational change from a coiled to a semi-helix-like state. This transformation, representing the lower critical solution temperature (LCST), enables the system to be converted into a semi-helix-like state upon heating. To examine the thermoresponsive behavior of CS and KC and the 3D printlets of E-5 and E-7 in water (Regarding CS in the dilute solution and acetic acid), a straightforward method involves turbidimetry measurements (Fig. 8a). The cloud point (Tc) for CS and KC was detected to be 42 and 33 °C. This temperature-sensitive property can serve as a useful indicator for assessing the complexation state of the 3D printlet and the hydrophobicity of the precursors.

Upon formation of the 3D printlet, a Tc of about 37 °C, is anticipated since the hydrophobic CNC or curcumin might be concealed by the hydrophilic CS and KC during L-PBF printing. This observation demonstrates that the thermoresponsive characteristics of the CS and KC remain intact within the 3D printlet, thereby enriching the thermoresponsivity in the properties of the synthesized 3D printed object. This revelation confirms that the 3D printlet changed from processing with L-PBF. The observed shift in Tc to about 37 °C signifies not only the dissociation of the CS/CNC and KC/CNC but also the apparent physical hydrogen linkage within the 3D structure during L-PBF. Consequently, the shift towards a higher Tc is observed because the CS and KC become more hydrophilic compared to the 3D printlet. Despite the less pronounced shifts in Tc, the findings still demonstrate the potential for combining thermoresponsivity in 3D printlet. Consequently, this 3D printlet can be considered a true stimuli-responsive system. This feature allows for switching the system between the unbound state, the semi-helix-like state, and the entangled state.

In this study, we acknowledge that while we measured turbidity to validate the thermoresponsive properties of the materials, it is important to recognize that these printlets are not ideal thermoresponsive materials. The data presented in Fig. 8 were obtained from “normalized transmittance” measurements. When compared to a benchmark thermoresponsive material such as poly(N-isopropyl acrylamide) (PNIPAAm), our results indicate relatively low transmittance rates, measuring 18 % for chitosan, 24 % for KC, 26 % for E5, 35 % for E7, and 94 % for PNIPAAm, which we previously assessed (Fig. S9).

The complex dissociation of 3D printlet upon heating can also be investigated using dynamic light scattering (DLS) as demonstrated in Fig. 8b. This comparison highlights the differences in responsiveness between the two printlets of E-5 and E-7 and emphasizes the importance of understanding their unique properties for tailoring their applications. Upon comparing the particle size distributions at 10 °C and after heating to 50 °C for 30 min, a significant shift from 7.1 nm to 3.9 nm was observed. This shift towards lower Dh (distribution width) might be attributed to the expulsion of the functional groups, resulting in smaller particles in the system. Upon cooling back to 10 °C, the initial star polymers reformed, and the particle size distribution returned to 6.7 nm. Interestingly, the outcomes from the heating treatment of the 3D printlet showed significant differences. In the case of E-5, only insignificant amounts of aggregates were formed, while E-7 led to the formation of aggregates and agglomerates. These contrasting results support the assumption that the formation of physical linkages drives the irreversible aggregate formation under the L-PBF printing process.

Considering the investigation of redox stimuli, cyclic voltammetry
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** (a) Turbidimetry measurements at a concentration of 1 mg mL⁻¹. (b) Temperature-responsive dissociation of 3D printlets, 50 °C (dash lines), and 10 °C (solid lines) measured via DLS at a concentration of 2 mg mL⁻¹. Evaluation of the redox stimulus through CV measured in 0.1 M LiCl at 20 mV s⁻¹, (c) at 25 °C and (d) at 40 °C.</figcaption></figure>

(CV) was employed to analyze the 3D printed printlet (Fig. 8c). In the case of E-5, the CV trace exhibits an oxidation peak at 0.61 V and a reduction peak at 0.40 V, indicating reversible redox processes across 30 cycles. Notably, the E-7 printlet demonstrates distinct CV responses. Specifically, no discernible oxidation or reduction peaks were observed. The low response in CV is a known phenomenon, typically manifesting as weak peak potential signals. Several potential explanations for the undetectable oxidation and reduction signals in the current system can be proposed [57,58]. One possible reason is the restricted diffusion of 3D printed parts, which may hinder the oxidation and reduction processes at the electrode surface. Another more significant factor can be the strong complexation between CS and KC, which might prevent them from interacting effectively with the electrode, particularly in the context of the present 3D printed printlets. To determine whether the missing signals result from complex formation, additional measurements were carried out using CS, KC, CNC, and curcumin. Upon incorporating a competing guest molecule with a higher complexation constant, the oxidation and reduction signals reappeared, supporting the hypothesis that the missing signals are due to complex formation (data not shown). The impact of temperature-induced complex dissociation was also evident in CV experiments, as shown in (Fig. 8c,d). Upon heating the sample to 40 °C, weak oxidation and reduction signals were detected that were not observed at ambient temperature due to complexation. This demonstrates that a 3D printlet can be triggered to transition from the bound to the unbound state by applying heat. Furthermore, the 3D printlet exhibited a gated response to both

temperature and redox treatment. Depending on the applied stimulus, a switch to the unbound state or the formation of aggregates can be achieved. Furthermore, at elevated temperatures (40 °C), the signals were observed, which can be attributed to the thermal dissociation of complexes at higher temperatures (Fig. 8d). These findings collectively suggest that complex formation within 3D printlets can be responsible for the undetectable oxidation and reduction signals in the initial study. However, in cyclic voltammetry (CV) measurements, no signals were detected at elevated temperatures above the Tc of the CS and KC, which might be attributed to some moieties being inaccessible in the semi-helix-like state (data not shown). In summary, the synthesized 3D printlet displays various thermoresponsive characteristics. Understanding and controlling these temperature-dependent properties is crucial for optimizing the performance and applications of this 3D printlet.

### 3.5.7. *In vitro* drug release

The curcumin release profiles of 3D printlets displayed a sustained drug release over 24 h, contrasting the rapid release of the free drug within 4 h (Fig. 9a). The printlets release of curcumin exhibited a composition-dependent biphasic pattern. In the first phase, there was a rapid release, with up to 55 % of the drug released within the first 4 h, except for E-1 printlets, which took 12 h for equivalent drug release. The second phase featured a slower drug release rate, with E-5 and E-7 printlets releasing about 75 % of curcumin after 6 h. The E-1 printlet continued to release the drug gradually, reaching 90 % after 18 h. Statistical analysis of drug release kinetics from the prepared
<figure><img src="image_12.png" /><figcaption>Fig. 9. (a) In vitro release showing the release of curcumin over 24 h from 3D printlets, and (b) $R^2$ values for fitting linear regression equations for drug release kinetics. (c) Accumulative release curves of 3D printlets at various temperatures. (d) Illustration of the step-by-step, on-demand release of curcumin with cycles of temperature change between 16 and 37 °C. (e) Hydrodynamic size changes of 3D printlets at various temperatures (from 15 to 60 °C) by DLS. (f) Proposed mechanism for thermo-responsive curcumin release from 3D printlets.</figcaption></figure>

thermosensitive printlets was conducted using various models (Fig. 9b). It was found that the release of curcumin from all formulations conformed best to the Higuchi model, indicating that drug release occurred through diffusion within the 3D printlets. This observation aligns with previous studies demonstrating that drug release from thermosensitive and other printlets followed the same mechanism [37,39]. Furthermore, according to the diffusion theory, the time taken for a drug molecule to traverse a specific distance is proportional to the square of that distance and inversely proportional to the diffusion coefficient. Consequently, drug diffusion through the monolithic matrix or aqueous pores is more efficient over shorter distances, with effectiveness decreasing over longer distances. This concept helps explain the observed biphasic release pattern of the drug from the prepared formula. Drug molecules near the surface of the 3D printlets were released more rapidly, while the release rate decreased for those located deeper within the printlets, as they gradually diffused to the surface and traveled longer distances.

The release of curcumin from 3D printlets was influenced by several factors, including porosity, swelling ratio, gelation time, and mechanical properties. More porous materials facilitate easier water permeation and faster drug diffusion due to their open structure. The printlets with higher swelling ratios provide a more hydrophilic microenvironment, promoting faster drug dissolution and diffusion. The sol-to-gel transition rate impacts drug entrapment efficiency within the hydrogel matrix; slower gelation allows the formulation to remain in the liquid state for a longer duration, enabling faster initial burst release of the drug. Lastly, the drug release rate is inversely proportional to mechanical properties, as higher tensile strength restricts water permeation and drug diffusion. In the case of E-1 printlets, its relatively lower swelling ratio, inferior porosity, along lower mechanical strength, contribute to the lack of control over the release of curcumin from the matrix. Expectedly, E-5 or E-7 resulted in higher sustained release compared to E-1 and E-3. This

can be attributed to the fact that these printlets, despite having a higher mechanical strength, exhibit a higher porosity. This allows for better water permeation and drug diffusion, consequently affecting the drug release profile.

### 3.5.8. Release of drug at various temperatures

The investigation focused on evaluating the temperature-dependent release of curcumin from 3D printlets at temperatures ranging from 16 to 37 °C. The results revealed that all samples offered a higher curcumin release with increasing temperature. Specifically, E-1 exhibited considerably slower release rates (data not shown) compared to E-7 (Fig. 9c). The E-7 also demonstrated a controlled release behavior in response to temperature variations, with the released quantities of curcumin showing an increase with higher temperatures. In this case, E-7 displayed a gradual release of curcumin (approximately 15% at 16 °C over a 24 h period), while E-1 exhibited a similarly low release percentage (around 11% period at 16 °C over a 24 h). Upon increasing the temperature to 25 °C, the E-7 printlet demonstrated a moderate release of curcumin (approximately 25% over 24 h), while E-1 released an accumulated quantity of 16.4% of curcumin. The release of curcumin notably increased at a higher temperature (37 °C), with accumulative quantities rapidly reaching about 85% for E-7, after 24 h. Next, an assessment of the on-demand release behavior of curcumin from 3D printlets (Fig. 9d) was conducted. It was observed that at 16 °C, the release curve remained constant, while at 37 °C, the release rate increased rapidly. The release plateaued once the temperature dropped back to 16 °C, and subsequent temperature increments to 37 °C saw a renewed increase in release rates. This thermally responsive release pattern aligns with the body temperature, which can be used in the field of smart release of bioactive compounds. The temperature-responsive release of curcumin from 3D printlets is likely connected to their
precursor (CS and KC) thermoresponsive polymers, which were sinter
during the L-PBF printing process. To investigate this release mecha-
nism, we conducted a quantitative analysis of the size of 3D printlets
using DLS across a temperature range of 15 to 60 °C (Fig. 9e). In this
case, a significant thermally responsive size variation behavior for E-7
was observed, illustrating the structural changes in the 3D object at the
evaluated temperatures. At a lower temperature (16 °C), the size of 3D
printlets was measured at 322.4 nm. As the temperature increased to
25 °C, the sizes reduced to 241.2 nm. Remarkably, at about 37 °C, the
sizes of the 3D printlets significantly decreased to 55.1 nm. Overall,
there was a substantial variation in size and volume for 3D printlets,
exceeding 200-fold, between 16 to 37 °C. These remarkable changes in
size and volume correspond closely to the release of curcumin triggered
by temperature changes, as illustrated in Fig. 9f.

4. Conclusion

The binary combinations of CNC-KC and CNC-CS coalesced into W/
W emulsions upon agitation through ultrasonication. Our findings pro-
vide a unique glimpse into the internal organization and colloidal as-
sembly of the all-aqueous bicontinuous LC emulsion. The analysis
revealed that the isotropic phase contains a three-dimensional, inter-
connected network of cholesteric LC, formed through the trapping and
self-assembly of CNC within binary polymer domains within the emul-
sion. The CNCs exhibited varying affinities for the binary CS and KC
phases due to the equilibrium of osmotic and chemical potential dif-
ferences between the two phases, resulting in the entrapment of CNCs
within the aqueous CS component rather than the KC component. The
aforementioned results suggest that CNC undergoes hierarchical self-
assembly within intricate polymer domains, ultimately forming a
bicontinuous structured LC emulsion with a cholesteric-in-isotropic
arrangement. As the CS and KC phases separate during their phase
transition, an abundance of CNC particles becomes encapsulated within
the CS domains. This leads to additional aggregation and self-assembly
of CNCs into a fingerprint cholesteric organization when the concen-
tration surpasses a critical threshold. Furthermore, an excess of CNCs
can autonomously self-organize into a cholesteric structure within the
KC phase, while CNCs in the CS phase remain in an isotropic state,
leading to the formation of a percolating bicontinuous structured LC
emulsion. This phenomenon demonstrates the particle segregation-
induced structural confinement of CNCs within one polymer domain.
In the complex coacervation, CS and CNC formed spherical microcap-
sules due to the complexation between oppositely charged species,
which led to the even distribution of the CNC/CS phase dispersed in the
continuous CNC/KC phase. This resulted in the creation of spherical
CNC/CS droplets. Using L-PBF 3D printing technology, we have devel-
oped a novel approach to create customized 3D printlets that can pre-
cisely control the drug release characteristics of various pharmaceutical
polymers. By adjusting parameters, we could tailor the drug release
behavior to meet individual patient needs. This breakthrough allows for
personalized medication performance, leveraging a single standardized
ink without the need for modifying the formulation composition,
thereby streamlining the process for patients with unique requirements.
The findings offer essential data for the formulation of drug crystalli-
zation encapsulated LC emulsion and the characterization of the
encapsulated drug's physical state within the LCs carrier systems. This
information is crucial for the development of innovative drug delivery
systems for drug delivery in pharmaceutical applications. Thus, it can be
concluded that the thermoresponsive 3D printlets as intelligent 4D
structures with an on-demand, controlled release and synergistic bio-
logical activity and, in so doing, prolong release duration and improve
the utilization efficiency.

CRediT authorship contribution statement

Mahdiyar Shahbazi: Writing – review & editing, Writing – original

draft, Software, Resources, Project administration, Methodology, Investigation, Data curation, Conceptualization. **Henry Jäger:** Writing – original draft, Supervision, Methodology, Investigation, Conceptualization. **Rammile Ettelaie:** Writing – original draft, Validation, Methodology, Investigation, Conceptualization. **Peyman Asghartabar Kashi:** Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. **Delphine Huc-Mathis:** Writing – original draft, Methodology, Investigation. **Jianshe Chen:** Writing – original draft, Validation, Methodology.

5. Funding

The research funding was provided by the University of Natural
Resources and Life Sciences Vienna (BOKU).

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cej.2024.157614.

Data availability

Data will be made available on request.

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