Optimization of the spherical integrity for sustained-release alginate microcarriers-encapsulated doxorubicin by the Taguchi method

Hepatocellular carcinoma (HCC) is commonly diagnosed at advanced stages. Transarterial chemoembolization (TACE) delivers drug-loaded microcarriers into the hepatic artery to induce embolization and hypoxia and is a standard, minimally invasive therapy for unresectable HCC. Existing FDA-approved commercial drug-eluting beads are effective but are expensive and non-biodegradable, which can lead to permanent embolization and related complications. Biodegradable, biocompatible polymers such as sodium alginate offer advantages as drug carriers due to their safety, hydrophilicity, and gelation via ion crosslinking with calcium. Prior studies show alginate systems can encapsulate drugs and modulate release, including doxorubicin (Dox) for delayed hepatic release. However, process parameters strongly affect bead morphology (spherical integrity), size, and performance; high alginate concentrations can form macrogels. This study aims to identify optimal fabrication parameters using the Taguchi method to produce calcium alginate microcarriers with uniform size and high spherical integrity that enable sustained Dox release suitable for TACE.

Modern embolic agents include temporary and permanent materials; commercial drug-loaded microspheres (e.g., HepaSphere, DC Bead) are non-biodegradable and costly. Biodegradable polymers (chitosan, gelatin, chitooligosaccharide, alginate) have been explored for drug delivery. Alginate, a GRAS polyanionic polysaccharide composed of M and G units, forms calcium alginate gels by ionic crosslinking and has been used to encapsulate peptides, antigens, and small molecules. Water-based ion crosslinking has produced alginate microspheres for drugs like pindolol and genipin. Dox-loaded alginate microspheres can prolong hepatic retention in vivo. Morphology and mechanical properties depend on formulation; higher alginate concentration can yield macrogels, and hydrogels often exhibit lower mechanical strength than solid polymers. Tumor microenvironments are acidic (Warburg effect), suggesting pH-responsive swelling and release may be advantageous for targeted delivery.

Design and optimization: A Taguchi L18 (2^1 × 3^7) orthogonal array was used to optimize spherical integrity. Eight control factors were varied at specified levels: A) cross-linking volume ratio (sodium alginate:CaCl₂) = 0.17, 0.1; B) sodium alginate concentration = 1.5, 2.0, 2.5 wt%; C) CaCl₂ concentration = 3, 6, 9 wt%; D) collection distance = 2, 5, 8 cm; E) flow rate = 30, 40, 50 mL/h; F) stirring speed = 100, 150, 200 rpm; G) syringe needle inner diameter = 0.25, 0.20, 0.15 mm; H) hardening time = 1, 2, 3 h. Eighteen formulations were prepared in triplicate. Spherical integrity scoring: Microcarriers were immersed in PBS for 14 days; appearance was scored 1–5 (1 = dissolved; 5 = intact) and converted to signal-to-noise (S/N) ratios for analysis of factor effects and ANOVA to determine contributions and optimal settings. Preparation of microcarriers: Sodium alginate solutions (1.5–2.5 wt%) were prepared in deionized water, stirred overnight, and degassed by ultrasonication. CaCl₂ solutions (3–9 wt%) were autoclaved. For Dox loading, Dox (2 mg/mL in the alginate solution) was mixed prior to extrusion. Using a syringe on an infusion pump, alginate (with or without Dox) was extruded dropwise through a gauge nozzle into sterile CaCl₂ baths at set collection distances. Ionic crosslinking formed calcium alginate beads. Beads were stirred (typically 150 rpm) for curing per assigned hardening times, filtered, washed twice with sterile water, and stored in 0.06 wt% CaCl₂ at room temperature. Characterization: FTIR (KBr pellets, 4000–400 cm⁻¹) was used to assess functional groups and confirm crosslinking for F1, F2, F3 vs sodium alginate. SEM imaged dried microcarriers (with and without Dox) to assess surface morphology. Swelling studies: Hydrogel-form beads (0.04 g) were immersed in PBS at 37 °C at pH 7.4 or 6.5; at intervals up to 240 min, beads were blotted and weighed to compute swelling rate (%) = (Wt − W0)/W0 × 100. Encapsulation and loading efficiencies: After preparing Dox-loaded beads, residual Dox in the syringe and gelation bath was quantified spectrophotometrically (NanoDrop 2000) at 230 nm using a calibration curve (0–0.4 mM). Encapsulation efficiency (%) = Wi/Wt × 100; Loading efficiency (%) = Wi/Wdm × 100, where Wi is Dox mass in beads, Wt initial Dox mass, Wdm dry bead mass. Release studies: Approximately 0.3 g beads were placed in hanging inserts over 1.5 mL PBS (pH 7.4) or PBS + 10% FBS at 37 °C. At intervals, supernatant was sampled (1 μL) and Dox quantified; cumulative release (%) = Wds/Wt × 100. Controls without Dox corrected background. In vitro anticancer activity: Huh-7 and Hep-3B HCC cell lines were cultured in DMEM + 10% FBS at 37 °C, 5% CO₂. Cells (seeded in 12-well plates) were co-cultured with inserts containing F1 or F3 beads with or without Dox, allowing released Dox to diffuse to cells. Viability was measured by trypan blue exclusion on days 4, 8, and 12. Media with cumulative Dox were refreshed every 4 days. Statistical analysis used one-way ANOVA with Scheffé post hoc tests (p < 0.05).

- Fabrication and morphology: Eighteen formulations produced microcarriers of ~2 mm size (range 1.1–1.6 mm) with varied shapes (irregular, oval, tear-drop). After 14 days in PBS, several groups (e.g., #5, #6, #14) maintained intact surfaces, while others became translucent or degraded. - Taguchi optimization: Factor response analysis identified collection distance (D) as most critical for spherical integrity (contribution ratio 48.839%), followed by hardening time (H, 16.395%) and CaCl₂ concentration (C, 11.416%). Optimal parameters (F1) for maximal spherical integrity were: A = 0.1; B = 2.5 wt%; C = 6 wt%; D = 8 cm; E = 30 mL/h; F = 150 rpm; G = 0.25 mm; H = 2 h. F2 represented intermediate, and F3 the worst integrity. - FTIR: Crosslinked beads (F1–F3) exhibited characteristic −OH (3000–3600 cm⁻¹) and −COO⁻ (∼1342, 1423, 1622 cm⁻¹) bands, and 935–1107 cm⁻¹ ring/−C−O stretches, with narrowed −OH bands vs sodium alginate, consistent with Ca²⁺ chelation and ionic crosslinking. - SEM morphology: Dox-loaded beads showed rougher, wrinkled surfaces vs blank beads; F3 had the densest wrinkles and greatest surface area, consistent with lower alginate concentration effects and potentially faster release. - pH-sensitive swelling: At pH 7.4, rapid swelling within 30 min: 97.88% ± 3.01 (F1), 108.88% ± 4.42 (F2), 110.13% ± 7.95 (F3). Maximal swelling at 240 min: F1 145.13% ± 1.24, F2 149.13% ± 12.90; F3 overexpanded and began disintegrating (reduced by 10.88% at 240 vs 180 min). At pH 6.5, swelling was reduced: 30-min values 45.25% ± 1.06 (F1), 47.38% ± 7.25 (F2), 53.25% ± 7.42 (F3); maximal at 240 min: 65.75% ± 2.83 (F1), 73.44% ± 0.35 (F2), 95.00% ± 12.72 (F3). - Encapsulation/loading: Encapsulation and loading efficiencies were highly correlated. F3 achieved highest encapsulation 48.05% ± 0.76 and loading 4.73% ± 0.25; F1 achieved ~40.62% ± 0.85 encapsulation and 3.52% ± 0.13 loading; F2 37.61% ± 0.71 and 3.11% ± 0.14. - In vitro release in PBS (pH 7.4, 37 °C): Slow release during first two weeks; cumulative by week 2: F1 0.15% ± 0.06, F2 0.51% ± 0.00, F3 0.33% ± 0.10. By day 38: F1 2.21% ± 0.02, F2 2.21% ± 0.01, F3 2.71% ± 0.06. F2 and F3 showed burst releases after ~day 25; F1 remained gradual. - Release in PBS + 10% FBS: Faster and bursty after day 12. Day 14 cumulative release: F1 10.35% ± 4.35, F2 19.33% ± 3.02, F3 14.20% ± 6.08. Experiments terminated at day 16 due to contamination/variability. - Cytocompatibility and anticancer effects: Blank calcium alginate beads were non-toxic. Huh-7 viability with F1/F3 blanks on day 4: 96.00% ± 2.00 / 97.67% ± 0.58; Hep-3B: 83.33% ± 5.69 / 79.00% ± 9.64. Dox-loaded F1 caused time-dependent decreases in Huh-7 viability: days 4/8/12 = 73.67% ± 5.57 / 47.00% ± 2.65 / 35.67% ± 2.68. Dox-loaded F3 reduced viability substantially by day 4 with similar levels through day 12. In Hep-3B, Dox-loaded F1 days 4/8/12 = 39.67% ± 2.52 / 39.00% ± 7.51 / 27.67% ± 1.73; Dox-loaded F3 days 4/8/12 = 36.67% ± 3.06 / 32.33% ± 4.62 / 27.67% ± 0.58. Overall, Dox-loaded beads reduced viability to ~30% by day 12 (~70% inhibition), with F1 exhibiting better sustained-release behavior.

Using a systematic Taguchi L18 design, the study identified key process parameters that govern spherical integrity of calcium alginate microcarriers, a critical quality attribute for embolization and controlled drug release. Collection distance, hardening time, and CaCl₂ concentration were the dominant factors, with the optimal set (F1) producing the most structurally stable, spherical beads. FTIR confirmed ionic crosslinking without altering the characteristic alginate chemistry. SEM revealed that drug incorporation increased surface roughness, particularly under suboptimal fabrication (F3), which likely enlarged surface area and contributed to burst release behavior. The swelling experiments demonstrated pH sensitivity: beads swelled less at pH 6.5 than at pH 7.4, aligning with the acidic tumor microenvironment (Warburg effect). Reduced swelling at tumor-relevant pH may enhance structural stability in situ and modulate release kinetics during TACE. Release tests showed that under physiological buffer, F1 provided the most gradual, sustained Dox release over weeks, while F2 and F3 exhibited late-stage bursts, consistent with their structural deficiencies. The presence of serum proteins (10% FBS) accelerated release and increased variability, indicating protein interactions or enzymatic effects can destabilize the hydrogel network. Biocompatibility was supported by high viabilities with blank beads. Dox-loaded beads effectively suppressed HCC cell viability over 12 days, with F1 showing a progressive, sustained cytotoxic effect consistent with controlled release, whereas F3 likely delivered an early higher dose. These findings support the hypothesis that optimizing spherical integrity via process parameters improves sustained-release performance and therapeutic potential for TACE.

The study developed biodegradable calcium alginate microcarriers optimized for spherical integrity and sustained doxorubicin release using a Taguchi L18 design. The optimal fabrication parameters were a cross-linking volume ratio (alginate:CaCl₂) of 0.1, 2.5 wt% alginate, 6 wt% CaCl₂, 8 cm collection distance, 30 mL/h flow rate, 150 rpm stirring, 0.25 mm needle, and 2 h hardening. Under these conditions (F1), beads exhibited superior structural stability, pH-sensitive swelling, gradual Dox release over weeks in PBS, and effective, sustained in vitro anticancer activity, while remaining non-toxic without drug. The work highlights key processing factors (collection distance, hardening time, CaCl₂ concentration) that most influence bead integrity and performance. Future research should evaluate in vivo TACE efficacy and safety, investigate serum/protein effects on release and stability, and further optimize the balance between encapsulation efficiency and sustained-release kinetics.

- Release studies in protein-containing media (PBS + 10% FBS) experienced contamination and variability after day 12–14, leading to early termination and large error bars, limiting interpretation of serum effects. - SEM observations were performed on dried beads; drying induced shrinkage and surface cracking, which may not reflect hydrogel-state morphology during use. - Mechanical properties of hydrogels are generally lower than solid polymers; no mechanical testing under flow/physiological conditions was reported. - All efficacy assessments were in vitro with two HCC cell lines; no in vivo embolization or pharmacokinetic/therapeutic evaluations were conducted. - Trade-offs between higher encapsulation/loading (F3) and sustained-release/structural integrity (F1) were observed; optimization toward both goals concurrently was not fully resolved.