<figure><img src="image_1.png" /></figure>

HAL
open science

# Degradable Hydrogel Microspheres for Drug Delivery:
In Vitro Performance and Influence of E-Beam
Sterilization

Laurent Bédouet, Anne Beilvert, Emeline Servais, Laurence Moine

► To cite this version:

Laurent Bédouet, Anne Beilvert, Emeline Servais, Laurence Moine. Degradable Hydrogel Microspheres for Drug Delivery: In Vitro Performance and Influence of E-Beam Sterilization. AAPS PharmSciTech, 2025, 26 (5), pp.162. 10.1208/s12249-025-03144-1. hal-05099904

HAL Id: hal-05099904

https://hal.science/hal-05099904v1

Submitted on 5 Jun 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Degradable Hydrogel Microspheres for Drug Delivery: *In Vitro* Performance and Influence of E-Beam Sterilization

Laurent Bédouet<fnref n="1" /> ID · Anne Beilvert<fnref n="1" /> · Emeline Servais<fnref n="1" /> · Laurence Moine<fnref n="2" />

Received: 20 March 2025 / Accepted: 5 May 2025

© The Author(s) 2025

## Abstract

The present work examines the *in vitro* drug delivery performance of degradable microspheres (DrugMic) composed of a crosslinked hydrogel of poly(ethylene glycol), with particular interest on the effect of e-beam sterilization on the stability of loaded active substances, i.e. niflumic acid (14% w/w), tadalafil (7% w/w), travoprost (0.2% w/w), buprenorphine (1.4% w/w), teicoplanin (6% w/w), and polymyxin B (5.6% w/w). Drug loading was performed on preformed microspheres degradable in 3 days, 1 or 2 weeks. Drugs were loaded onto microspheres (50–100 µm or 500–700 µm) via 1 h room-temperature incubation. After freeze-drying, drug-loaded microspheres were sterilized using e-beam irradiation (15 or 25 kGy). *In vitro* drug releases were done in PBS, drug elution profiles and radiostability were assessed by RP-HPLC with diode array detection. Following irradiation, DrugMic delivered niflumic acid and tadalafil for 3 days, teicoplanin and polymyxin B for 1 week, buprenorphine and travoprost for 2 weeks from microspheres degradable in 3 days, 1 and 2 weeks. Radiolysis was observed in each fraction collected during buprenorphine (0.7–20%) and travoprost (1.8–139%) release, while the HPLC profiles of polymyxin B were completely altered, indicating substantial degradation. Niflumic acid, tadalafil and teicoplanin showed no signs of radiolysis. Extemporaneous loading of buprenorphine and polymyxin B onto sterilized microspheres was attempted to avoid radiolysis. The sustained release profiles were maintained without degrading the drug. DrugMic appears to be a suitable platform for sustained drug release. The loading mode can be adapted according to the stability of the drug to irradiation.

**Keywords** degradable microsphere · drug delivery · e-beam sterilization · hydrogel · radiolysis

## Introduction

To be used in patients, drug delivery systems (DDS) must meet sterility requirements [1, 2]. Sterility can be achieved through two main approaches: aseptic processing or terminal sterilization [3]. Aseptic processing is complex, requiring meticulous precautions to prevent contamination throughout the manufacturing process. In contrast, terminal sterilization is more cost-effective and operationally efficient. However, the use of degradable drug carriers and the fragility of encapsulated drugs limit the choice of sterilization methods. Steam sterilization is not suitable for degradable DDS due to the hydrolytic degradation of the polymers, and ethylene

oxide sterilization leaves toxic residues that are difficult to remove. Therefore, excluding these sterilization methods, gamma and electron beam irradiation methods remain viable options. However, there are challenges associated with these irradiation methods. During irradiation, free radicals are formed in the excited polymer material which can have a life span of 14 days and induce undesirable chemical changes in both the polymers and the active pharmaceutical substances [2, 4].

The effect of irradiation on polymers is generally divided into chain scission (degradation at low irradiation dose) and crosslinking (polymerization at high irradiation dose). Chain scission along the polymer chains could accelerate the degradation of the microspheres, thereby accelerating drug release [5–7]. However, sterilization of degradable poly(lactic-co-glycolic) acid (PLGA)-based DDS does not necessarily result in accelerated release of encapsulated drugs [8–10]. The sensitivity to radiation is dependent on the microstructure and the composition of the polymers [2].

<footnote>Laurence Moine
laurence.moine@universite-paris-saclay.fr</footnote>

<footnote n="1">1. Occlugel, 17 Bis, Avenue Des Andes, 91940 Les Ulis, France</footnote>

<footnote n="2">2. Université Paris-Saclay, CNRS, Institut Galien Paris-Saclay, 17-19, Avenue Des Sciences, 91400 Orsay, France</footnote>
Another consequence of radiosterilization is the risk of damage to the encapsulated active substances [11]. The formation of degradation products during terminal sterilization is problematic because if these products are above the so-called "impurity" thresholds, demonstration of their safety must be performed by the manufacturer. To guide the experimenter in the choice of encapsulable active substances, the effects of ionizing radiations on around 400 pharmaceutical substances were published [12, 13]. However, many molecules are missing from these databases and their stability with respect to irradiation must be studied on a case-by-case basis. In the development of a DDS, the stability of the active substance with respect to sterilization is of fundamental importance. This parameter can be considered as limiting in the development process of a DDS.

This study investigates the radiostability of 6 pharmacologically relevant compounds incorporated into DDS: two antibiotics (polymyxin B and teicoplanin) efficient against multi-resistant bacteria; an opioid (buprenorphine), an non-steroidal anti-inflammatory drugs (NSAID) (niflumic acid), a prostaglandin analogue used for glaucoma treatment (travoprost), and a phosphodiesterase-5 inhibitor (tadalafil) with the potential to treat many human diseases including cardiovascular diseases and neurodegenerative disorders far beyond the treatment of erectile function and benign prostatic hyperplasia [14]. These compounds were selected since their encapsulation in a DDS can address a number of key issues that are often encountered: promoting drug adherence [15], minimizing the risk of antimicrobial resistance [16, 17], reducing dosing frequency [18, 19] and/or making treatments for chronic diseases such as glaucoma [20, 21] and hypertension [22] more effective and less burdensome. They were loaded onto prefabricated degradable microspheres known as DrugMic prior to sterilization and in vitro drug release. These hydrophilic degradable microspheres, initially developed for embolization and chemoembolization [23–27] are composed of a crosslinked hydrogel of poly(ethylene glycol) and degrade via hydrolysis.

Electron irradiation was selected as the sterilization method due to its ability to generate approximately half the amount of radicals in PLGA microspheres compared to gamma sterilization [28]. To evaluate the impact of this sterilization approach, drug loading was first performed by mixing the active substances with the manufactured microspheres, then the drug-loaded microspheres were lyophilized and sterilized by electron beam irradiation at 15 kGy or 25 kGy. The in vitro release of the active substances was carried out in PBS and the fractions were analyzed by RP-HPLC coupled to a diode array detector (DAD). The effects of sterilization on the drug-free microspheres were evaluated in terms of size, morphology and degradation time. Appearance of new peaks on the chromatograms was considered as an evidence of the radiolysis of the active substances. In

the case of radiolysis of an active substance, extemporaneous loading for 5–15 min was attempted on sterilized drug-free microspheres, as in the case of chemoembolization, to evaluate drug delivery using this strategy, which is known to protect the active substance drug from degradation.

# Material and Methods

## Materials and Equipment

Poly(ethylene glycol) methyl ether methacrylate (PEGma) (Mn = 300 g/mol), poly(ethylene glycol) (Mn = 600 g/mol), ε-caprolactone, stannous 2-ethylhexanoate, methacrylic anhydride, methacrylic acid (MA), PBS, 2,2'-azobis(2-methylpropionitrile), buprenorphine hydrochloride, teicoplanin, travoprost, tadalafil, and niflumic acid were purchased from Sigma-Aldrich. DL-lactide was purchased from Polysciences. Polymyxin B sulfate was purchased from Merck. Chromatographic analyzes were carried out with the High-Performance Liquid Chromatograph Chromaster system (Merck-Hitachi, VWR International S.A.S). Acetonitrile (HPLC grade), trifluoroacetic acid (TFA) and dimethylsulfoxide (DMSO) were purchased from Carlo Erba. HPLC columns (C₄ and C₁₈) were purchased from Advanced Chromatography Technologies Ltd (ACE).

## Preparation of DrugMic

Poly(ethylene glycol) based degradable microspheres for drug delivery (DrugMic) were synthetized by suspension polymerization [23, 29, 30]. Briefly, 2 degradable crosslinkers (PEG₁₃-PLA₁₂ and PEG₁₃-PLA₇-co-PCI₃) have been synthesized by polymerization of poly(ethylene glycol), with DL-lactide and ε-caprolactone with stannous 2-ethylhexanoate during 20 h at 115°C. After polymerization and precipitation, the end-chain functionalization was done with methacrylic anhydride at 80°C for 8 h. Polymerization suspension was done with degradable crosslinkers, poly(ethylene glycol) methyl ether methacrylate (PEGma) and methacrylic acid at 80°C during 8 h using 2,2'-azobis(2-methylpropionitrile) as initiator. Three compositions of microspheres were prepared crosslinked at 5 mol% with crosslinker PEG₁₃-PLA₁₂ in size range 500–700 µm (DMic-1), PEG₁₃-PLA₇-co-PCI₃ in size range 50–100 µm without methacrylic acid (DMic-2) or with 30 mol% methacrylic acid (MA) for anionic loading of drugs (DMic-3). The composition of the microspheres used for the drug loading are summarized in table S1. Then, microspheres were collected by filtration on a 40 µm sieve and washed extensively with acetone and water. Microspheres were then sieved with decreasing size of sieves (900, 710, 500, 315, 100 and 50

<figure><img src="image_3.png" /></figure>
µm). The fractions 500–700 µm and 50–100 µm were collected for drug loading experiments.

## Optical Sizing

After the sieving step, wet microspheres were poured into a Petri dish and pictures were taken with an optical microscope at 2.5 × or 10 × magnification (Leitz, Diaplan) equipped with a digital camera (Allied Vision Mako). Microsphere diameter was determined with the Image J software (≈100 measures/condition) [31]. After sterilization, microspheres were suspended in saline during 10 min before analysis.

## Dried Weight Determination

In triplicate, after the sieving step, 1 mL of wet microspheres was placed in pre-weighted 15 mL polypropylene vials before freeze-drying (Christ, Alpha 2–4 LSC basic) at 0.2 mbar and –80°C during 2 days. Then, the dry microspheres weight (mg) was determined by weighing.

## In Vitro Degradation

Degradation experiments of microspheres were conducted in PBS at 37°C under shaking (150 rpm) as previously described [23]. The weight loss of microspheres was determined during the incubation time.

## Drug Loading on DrugMic after Synthesis before e-beam Sterilization

All drug loading experiments were performed at room temperature for 1 h on microsphere pellets (50–100 µm or 500–700 µm size ranges) collected after the sieving step in colorless and high-quality sterile conical tubes (15 mL) (Thermo Scientific™ Nunc) under stirring on a tube rotator (≈ 30 rpm).

## Niflumic Acid

1 mL of DMic-1 was suspended in 9 mL of a water/DMSO (3/6) mixture before the addition of 1 mL of niflumic acid (20 mg/mL) in DMSO prior to mixing. The determination of drug loading was determined by UV spectroscopy at 280 nm by measuring the residual amount of niflumic acid in the supernatant using a standard curve (0.78 to 50 µg/mL). Then, supernatant was discarded and the pellets were washed twice with water (10 mL).

## Tadalafil

DMic-2 (0.5 mL) was suspended in 8 mL of a water/DMSO (2/6) mixture before addition of 1 mL of tadalafil (20 mg/

mL) in DMSO prior to mixing. Tadalafil loading onto DMic-2 was determined by UV spectroscopy at 254 nm by measuring the residual amount of tadalafil in the supernatant using a standard curve (3.12 to 100 µg/mL). Then, supernatant was discarded, and the pellets were washed twice with water (10 mL).

## Buprenorphine

Buprenorphine hydrochloride (2 mg or 4 mg) in solution in 3 mL of water was added to 1 mL of DMic-2 pellets prior to mixing. Then, the supernatant was removed for the measurement of unbound buprenorphine by fluorimetry ($λ_{ex}$ 280 nm, $λ_{Em}$ 354 nm, Hitachi F2000). The amount of buprenorphine hydrochloride in supernatant was obtained by extrapolation from a standard curve (1.56 to 25 µg/mL). For the sterilization attempt at 15 kGy in order to reduce the radiolysis, 4 mg of buprenorphine were added to 1 mL of DMic-2. The buprenorphine-loaded microspheres were then suspended in an excipient solution (5% w/v glucose) prior to lyophilization with the goal of scavenging the reactive species, as has been described for other drugs [32, 33].

## Travoprost

To 250 µL of wet DMic-2 pellets, 1 mL of water was added, before the addition of 1 mL of travoprost solution (0.499 µg/mL in water/acetonitrile (70/30)) prior to mixing. Then, the supernatant (20 µL) was removed for the measurement of unbound travoprost by RP-HPLC as described in table S2 at 222 nm during isocratic separation in acetonitrile/water containing 0.1% TFA (60:40, v/v) at 25°C. The amount of travoprost in supernatant was obtained by extrapolation from a standard curve (0.05 to 10 µg/mL), and the loaded dose was calculated by subtraction.

## Teicoplanin

Pellets of 0.5 mL of DMic-3 were mixed with 1 mL of teicoplanin (10 mg/mL in water). The determination of teicoplanin loading was determined by UV spectroscopy at 280 nm by measuring the residual amount of teicoplanin in the supernatant with a standard curve (15.6 to 500 µg/mL) and the loaded dose was calculated by subtraction.

## Polymyxin B

Pellets of 0.5 mL of DMic-3 were mixed with 2 mL of polymyxin B (10 mg/mL in water). The determination of drug loading was determined by UV spectroscopy at 220 nm by measuring the residual amount of polymyxin B in the supernatant. The amount of polymyxin B in supernatant was
obtained by extrapolation from a standard curve (12.5 to 500 µg/mL), and the loaded dose was calculated by subtraction.

## Validation of Active Substances Assay Methods

The analytical methods used to quantify each active substance were validated in terms of linearity, precision and accuracy (Table S3).

## Determination of Drug Loading and Loading Efficiency

The amount of active substances loaded onto DrugMic was given in percentage of dry weight (w/w): Drug loading = (mg of active substance loaded onto 1 mL of microsphere) / (dry weight (mg) of 1 mL of drug-free microsphere) × 100, and also in mg per mL (mg/mL) as is common in chemoembolization for the quantification of anticancer agents loaded onto the drug eluting beads (DEBs) [34, 35]. The drug loading for 1 mL of microspheres was calculated by subtracting the unbound amount of active substances in supernatant from the initial amount added to the microspheres. The drug load on 0.25 mL and 0.5 mL was multiplied by 4 and 2 to obtain the drug load for 1 mL of microspheres. The loading efficiency was calculated by the following equation: Loading efficiency = (active substance in feed – active substance in supernatant)/active substance in feed) × 100.

## Freeze-Drying and Irradiation

The drug-loaded microspheres were frozen at –80°C overnight before freeze-drying (Christ, Alpha 2-4 LSC basic) at 0.2 mbar and –80°C during 4–5 days in the dark. Then, samples were stored at –80°C before terminal sterilization. Samples were exposed to e-beam radiation at room temperature and air atmosphere in a linear electron accelerator. The electrons emitted have a single energy level of 9.8 MeV ± 0.2 MeV, (34 kW) and the passage speed was adjusted to deliver a dose of 15 kGy or 25 kGy (Ionisos, Chaumesnil, France). The standard irradiation dose was set at 25 kGy according to the Ph. Eur. 5.1.1 guideline for sterilization of pharmaceuticals. In an attempt to reduce radiolysis, buprenorphine-loaded microspheres were treated at 15 kGy. During the same run, samples of teicoplanin and polymyxin B-loaded microspheres were also exposed to 15 kGy irradiation.

## In Vitro Release

The drug release experiments after sterilization were done in PBS (10 mM phosphate buffered saline; NaCl 0,136 M; KCl 0.0027 M; pH 7.4) at 37°C under shaking (150 rpm). At each sampling time, the medium was completely renewed

with fresh PBS (2 mL for travoprost, 14 mL for teicoplanin and polymyxin B, 25 mL for buprenorphine, 40 mL for niflumic acid and 400 mL for tadalafil). Sterile drug-free microspheres (Bland DrugMic) were incubated under the same conditions to provide fractions for RP-HPLC analysis as controls.

## Quantification of Drug Release

Drug release in PBS was measured using UV spectrophotometry at 280 nm for niflumic acid and fluorimetry (λex 280 nm, λEm 354 nm, Hitachi F2000) for buprenorphine under the same conditions used to assess drug loading. The other four compounds in the release fractions were quantified by RP-HPLC at a flow rate of 1 mL/min at 222 nm for travoprost, 205 nm for polymyxin, and 280 nm for teicoplanin as summarized in Table S2. The HPLC system was the Chromaster analytical system consisting of a quaternary pump (5160), an autosampler set at 20°C (5260), a column oven set at 25°C (5310) and a diode array detector (5430) (Merck-Hitachi). The control of the system, data acquisition and analysis were conducted with Chromaster System Manager (CSM) software (Merck-Hitachi).

## RP-HPLC-DAD for Identification of Degradation Products

Degradation products of drug substances induced by e-beam sterilization of the drug-loaded microspheres were identified and quantified by RP-HPLC on chromatograms obtained for each fraction collected during the drug release experiments (Table S2). In the PBS fractions, the area of each new peak generated after e-beam irradiation was measured and summed, then the total area of the degradation products was divided by the peak area of the drug to obtain a percentage of degradation. Sterile and drug-free microspheres were used as control for RP-HPLC analysis in order to not confuse the degradation products of the microspheres with the decomposition products of the active substances. From the UV spectrum collected during HPLC analysis of the release fractions, multiple wavelengths were investigated to detect peaks other than the parent drug and microsphere degradation products. Chromatograms were selected every 10 nm between 200 and 280 nm to identify formation of new peaks after sterilization. Purity peak index determination was performed to investigate whether the drug chromatogram peak was representative of a single entity [13]. For the calculation of the purity index, two spectra at 10% of the peak height at the beginning and at the end of the peak of interest were selected and compared; the significance threshold for the comparison of the spectra was set to 95%.
## Extemporaneous drug loading on Sterile Microspheres

After sieving, 0.5 mL or 1 mL of microspheres was suspended in 14 mL of a 2.5% (w/v) mannitol solution. After homogenisation, microspheres were collected, lyophilized and sterilized by e-beam irradiation (25 kGy, Ionisos, Chauvesnil, France).

**Polymyxin B loading:** to the dry and sterile pellet of DMic-3 (0.5 mL), 2 mL polymyxin B (10 mg/mL in water) was added and loaded at room temperature for 15 min under stirring (≈30 rpm). Supernatant absorbance was measured at 220 nm and polymyxin B load was determined as described above. Upon completion of loading, *in vitro* release was performed in 14 mL PBS (37°C, 150 rpm) and the medium was completely changed at each sampling time. The quantity of polymyxin B in the fractions was determined by RP-HPLC (Table S2).

**Buprenorphine loading:** 4 mL of buprenorphine (1 mg/mL in water) were added to 1 mL of dry and sterile pellet of DMic-2 before 5 min of mixing (≈30 rpm). The loading values were determined as described above prior to the start of drug release in 25 mL PBS (37°C, 150 rpm). Buprenorphine release was quantified by RP-HPLC using the mobile phase described in Table S2, but with a modified elution gradient resulting in a shorter elution time for buprenorphine: solvent B from 40 to 70% in 8 min before equilibrating for 4 min in 60% of solvent A.

## Statistics

Statistical analyses were performed on StatView SAS 2000 (SAS institute, Cary, NC). For comparison of two groups, non-parametric Mann-Whitney (MW) test was used. Significance was set at $p < 0.05$.

## Results and Discussion

### Effects of e-beam Sterilization on Unloaded Degradable Microspheres

DMic-1 was synthesized for uterine fibroid embolization (a minimally invasive procedure used in interventional radiology to remove uterine fibroids) with calibrated degradable microspheres of size range 500–700 µm. The local release of a NSAID, for instance niflumic acid, was proposed to reduce the pain triggered by the post-embolization syndrome through its local delivery in ischemic fibroids [36–38]. Sterilization at 25 kGy of the drug-free freeze-dried DMic-1 induced a small size reduction (12%) without altering their shape (Fig. 1). Sterile microspheres did not aggregate after re-hydration in saline. The degradation time in PBS was

longer after sterilization, 3 days instead of 2 days after synthesis.

DMic-2 of size range 50–100 µm was dedicated to general drug delivery applications: buprenorphine for pain relief, travoprost for local therapy of glaucoma, and tadalafil for diseases related with dysregulation of nitric oxide–cGMP–protein kinase G signalling [14, 39]. DMic-2 sterilization at 25 kGy did not change their aspect nor their size compared to the sieving step done after microsphere synthesis (Fig. 2). Morphologically, the microspheres were similar regardless the treatment, they have a smooth surface without defects. Sterilization did not change the diameter of the microspheres (≈90 µm) compared to the sieving step. The degradation time in PBS was not profoundly changed by e-beam irradiation at 25 kGy. A small but significant higher weight loss was measured at 1 week ($p = 0.033$) after sterilization compared to the sieving step. Microspheres degraded after 2 weeks in both conditions. Sterilization had little effect on properties of DMic platform of size range 50–100 µm degradable in 2 weeks. Sterilization of DMic-3 did not alter the appearance of the microspheres or their diameter compared to sieving as for the other 2 compositions. Sterilized microspheres degraded *in vitro* within 1 week (Figure S1).

The drug-free degradable microspheres were not profoundly altered after sterilization at 25 kGy. The mild contraction of DMic-1 after irradiation could be explained by formation of additional crosslinks which is in line with the slower degradation rate. On the contrary, DMic-2 did not contract after sterilization nor degrade slower. These differences could be explained by the crosslinker composition, block copolymers of PLA-PEG-PLA for DMic-1, and block copolymers of lactide and caprolactone for DMic-2. According to the property of lactide units to increase the radical content in PLGA microspheres irradiated by e-beam [28], less radical would be produced in DMic-2 than in DMic-1, leading to less crosslinking after irradiation.

### Drug Loading on DrugMic and Drug Delivery after Sterilization

The amounts of active substances loaded on the degradable microspheres are summarized in Table I. The mixing of the various active substances with the degradable microspheres led to efficient drug loading, whatever the chemical structure of the active substance. This way of preparing DDS differs from the encapsulation of drugs in PLGA-based microspheres where active substances and polymers are mixed together before microsphere manufacturing [11, 20, 40]. The approach investigated in this study was inspired by chemoembolization with drug eluting beads (DEBs) used for the local delivery of anti-cancer drugs [34, 35]. This procedure aims to preserve the active substances by avoiding contact
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Effects of e-beam sterilization (25 kGy) on DMic-1 (500–700 µm). Aspect of microspheres after sieving (A), and sterilization after hydration of microspheres in saline (B), diameter values (C) and *in vitro* degradation time (D). Two batches were analyzed</figcaption></figure>

with organic solvents, high temperatures and mechanical shear stress used for encapsulation in PLGA microspheres.

After e-beam sterilization the release of the various drug-loaded microspheres in PBS was investigated (Fig. 3). Niflumic acid release lasted up to 3 days until DMic-1 (500–700 µm) degraded as shown in Fig. 1D. After one day, 50% of niflumic acid was released. Tadalafil loaded onto DMic-2 (50–100 µm) was fast eluted, 60% in 24 h, before reaching a plateau after 3 days, while buprenorphine and travoprost had a gradual release without a significant burst in about two weeks. Degradation of DMic-2 microspheres took place over 2 weeks, as shown in Fig. 2D. A sustained release of teicoplanin and polymyxin B was achieved during the degradation of DMic-3 in one week. The release of the 2 antibiotics took place during the degradation of DMic-3 microspheres over a one-week period (Figure S1D). This ability to load various active substances on the DrugMic microspheres enabled a controlled and sustained drug release, in phase with the degradation of the microspheres.

DrugMic degradation time increased with the crosslinker content in caprolactone instead of lactide (DMic 1 vs. DMic 2), while methacrylic acid accelerated microsphere degradation (DMic 3 vs. DMic 2). Caprolactone retards polymer degradation due to its hydrophobicity [41, 42] and carboxylate groups increase water diffusion into polymer network, which accelerates crosslinker hydrolysis and microsphere degradation [23]. During hydrolysis in PBS, microspheres remained spherical without fragmentation into debris, and remained visible until the final stages of degradation before disappearing (Figure S2). An outer shell was the last to hydrolyze, at a time when mass loss was already significant (80%) (Fig. 2). For the different crosslinked hydrogel formulations, the same behavior was observed: roundness shape maintained, no fragmentation, and swelling (≈ 40%) at the end of degradation. The degradation times of DrugMic would allow for a short drug release time, which is not the case with PLGA microspheres, which degrade over a period of months [43, 44].
<figure><img src="image_5.png" /><figcaption>**Fig. 2** Effects of e-beam sterilization (25 kGy) on DMic-2 (50–100 µm). Aspect of microspheres after sieving (A), after sterilization and hydration of microspheres in saline (B), diameter values (C) and *in vitro* degradation in PBS (37°C) (D). Four batches were analyzed</figcaption></figure>

## Effects of e-beam Irradiation on Active Substances Loaded on the Degradable Microspheres

The impact of terminal sterilization on the radiostability of each drug loaded on the degradable microspheres was analyzed by RP-HPLC-DAD in the fractions collected during the drug release experiments. The effects of e-beam irradiation at 15 kGy and 25 kGy were studied using two approaches: firstly, by measuring the purity peak index of the active substances detected in the release fractions and, secondly, by analysing individual chromatograms to identify the appearance of new peaks.

## Purity Peak Analysis of Active Substances after e-beam Treatment Using Diode-Array Spectral Data

The purity analysis of the chromatographic peak of the active substances found in each of the all-time release

fractions was determined using the UV spectra recorded during RP-HPLC fractionation (Table I). Compared to the drug standards used to generate the calibration curves, the peak purity values for five of the six drugs released from DrugMic did not differ significantly. Specifically, the spectral similarity between the start and end points of the chromatographic peaks for niflumic acid, tadalafil, buprenorphine, travoprost, and teicoplanin in the release fractions exceeded 95% after e-beam treatment. The high degree of spectral similarity suggests that the chromatographic peaks contained the active substance as unique chemical entity. However, the possibility of co-eluting radiolysis degradation products cannot be entirely excluded, particularly if such impurities were present at lower concentrations than the native compound and exhibited similar spectral characteristics [13]. Polymyxin B could not be analysed in terms of peak purity because of the profound alteration of its chromatographic profile after sterilization of the drug-loaded microspheres (Fig. 4A).
<table><caption>**Table 1** Loading values of active substances on preformed DrugMic and the effects of e-beam sterilization on the purity peak index of active substances released in PBS</caption><thead><tr><th></th><th>Microspheres</th><th>Drug loading (w/w)</th><th>Loading efficiency %</th><th>E-beam treatment</th><th>Retention time (min) of active substances eluted after sterilization*</th><th>Purity peak index of standards**</th><th>Purity peak index of active substances after DMic sterilization*</th></tr></thead><tbody><tr><td>Niflumic acid</td><td>DMic-1</td><td>14.4% (n=6)</td><td>72% (14.4 mg/mL)<sup>a</sup></td><td>25 kGy</td><td>5.28 (42)</td><td>0.9859<br>Min: 0.9646<br>Max: 0.9999</td><td>0.9813<br>Min: 0.9617<br>Max: 0.9984</td></tr><tr><td>Tadalafil</td><td>DMic-2</td><td>8.7% (n=4)</td><td>46% (18.7 mg/mL)</td><td>25 kGy</td><td>10.38 (17)</td><td>0.9926<br>Min: 0.9804<br>Max: 0.9977</td><td>0.9955<br>Min: 0.9687<br>Max: 0.9998</td></tr><tr><td>Buprenorphine</td><td></td><td>0.7% (n=2)<br>1.4% (n=5)</td><td>79% (1.5 mg/mL)<br>78% (3.1 mg/mL)</td><td>15 kGy, 25 kGy</td><td>8.60 (43)</td><td>0.9998<br>Min: 0.9998<br>Max: 0.9999</td><td>0.9998<br>Min: 0.9997<br>Max: 0.9999</td></tr><tr><td>Travoprost</td><td></td><td>0.21% (n=2)</td><td>90% (0.45 mg/mL)</td><td>25 kGy</td><td>6.09 (32)</td><td>0.9996<br>Min: 0.9986<br>Max: 1</td><td>0.9998<br>Min: 0.9986<br>Max: 1</td></tr><tr><td>Polymyxin B</td><td>DMic-3</td><td>5.6% (n=4)</td><td>36% (14.4 mg/mL)</td><td>15 kGy</td><td>Peaks eluted between 7.3–8.5 (16)</td><td>NA</td><td>NA</td></tr><tr><td>Teicoplanin<sup>b</sup></td><td></td><td>6% (n=4)</td><td>77% (15.5 mg/mL)</td><td>15 kGy</td><td>7.45 (24)</td><td>0.9999<br>Min: 0.9999<br>Max: 1</td><td>0.9999<br>Min: 0.9999<br>Max: 1</td></tr></tbody></table>

Loading values are expressed in weight (w/w) and in mg of drug for 1 mL of wet microsphere pellet (mg/mL) as usually used in chemoembolization [34, 35]. * values as mean determined from fractions collected during the release experiments in PBS after sterilization of drug-loaded DMic, (xx): number of chromatograms analysed, ** values as mean determined from standard curves used for HPLC assay of each drug (5 to 8 concentrations of standard). $: the main peak of the teicoplanin mixture eluted between 6.5 and 8.5 min. NA: not applicable (mixture of peptides)

# Analysis of the RP-HPLC Chromatograms to Investigate Radiolysis Degradation Products

## Active Substances Altered by e-beam Sterilization

**Polymyxin B:** sterilization at 15 kGy did not cause any yellowing of the polymyxin B loaded microspheres. Prior to sterilization, polymyxin B standard appeared as a chromatographic massif composed of 2 main peaks (Fig. 4A), presumably polymyxin B1 and polymyxin B2, since polymyxin B is a lipopeptide antibiotic mixture derived from *Paenibacillus polymyxa* in which more than 30 distinct polypeptides have been identified [45, 46]. After sterilization, a profound change in the appearance of the polymyxin profile was observed by RP-HPLC analysis at 205 nm of fractions collected after release in PBS (Fig. 4A). Instead of a massif consisting of 2 main peaks, sterilization produced a wider massif consisting of 3 peaks of roughly equal height. Additional peaks eluted between the injection peak and the polymyxin B massif were not detected in the multi-wavelength analysis. Alteration of the peaks profile eluted between 7 min and 8.5 min after sterilization of loaded DMic-3 suggests degradation of polymyxin B isoforms. The stability of

peptides appears to be variable with respect to sterilization. For example, gamma sterilization at 25 kGy has no effect on insulin but causes a 20% degradation of glucagon [13]. The observed data on polymyxin B degradation align partially with the findings compiled by Boess and Bögl (1996), which reported 14% and 32% decomposition of polymyxin B after gamma sterilization at 25 kGy and 250 kGy, respectively, while gamma sterilization at 100 kGy did not reduce the antibiotic activity of polymyxin E in its dry state [12]. The combination of these data, which were collected at different times [47, 48] and by different methods, suggests that polymyxin B is sensitive to sterilizing radiations (gamma and beta), even at low energy level of 15 kGy.

To overcome the radiolysis barrier, polymyxin B was loaded on pre-sterilized DMic-3 through rapid mixing for 15 min (Figure S3 A). Subsequent immersion in PBS induced a sustained release similar to that observed for sterilized polymyxin B loaded-microspheres (Figure S3B). Chromatographic analysis of the release fractions collected after extemporaneous loading onto DMic-3 revealed unaltered polymyxin B profiles (Fig. 4B), with the two major peaks corresponding to those of the polymyxin B standard, in contrast to the microspheres loaded and sterilized prior to
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>
►Fig. 3 *In vitro* drug release profiles in PBS of DrugMic after e-beam sterilization. Drug-loaded microspheres were incubated in PBS at 37°C under shaking for the release of niflumic acid (n = 3) (A), tada-lafil (n = 3) (B), buprenorphine (n = 3) (C), travoprost (n = 2) (D), teicoplanin (n = 2) (E) and polymyxin B (n = 2) (F). The medium was completely renewed at each time point. † Degradation time of microspheres during the drug release experiments

release (Fig. 4A). The DrugMic platform enables the sus-
tained release of polymyxin B through rapid loading onto
pre-sterilized microspheres, as practiced in chemoemboliza-
tion. This strategy avoids the need of final sterilization after
drug loading, thereby preserving the integrity of polymyxin
B.

**Buprenorphine:** sterilization at 25 kGy of the buprenor-
phine loaded microspheres resulted in yellow colored micro-
spheres. Color intensity increased with buprenorphine dose
(data not shown). RP-HPLC analyses showed that steriliza-
tion caused partial degradation of buprenorphine (Fig. 5A),
with the appearance of new peaks that did not correspond
to DMic-2 degradation products released in PBS during the
drug release. Five secondary peaks were identified as radi-
olysis products after 10 min and 5 h of release in PBS. Radi-
olysis was quantified at each time point, and surface of each
degradation product was summed for comparison to that of
the buprenorphine peak. Buprenorphine degradation peaks
were present at each sampling time, from 10 min to 12 days
(Fig. 6A). The area of the degradation peaks represented
between 0.7% and 20% of the surface of the intact buprenor-
phine chromatogram peak. The elution of degradation

products was biphasic, an important elution occurred imme-
diately after re-hydration of sterile microspheres followed by
a sustained release during the degradation of microspheres
and the concomitant release of buprenorphine.

The correlation of the UV spectra of the degradation products with buprenorphine UV spectra was variable, suggesting a close relationship with buprenorphine for products 4 and 5, and few structural similarity for the 3 other degradation products (Fig. 6B). Other degradation products could exist and be undetectable to UV detector, such volatiles derivatives or by-products strongly bound to the C₁₈ column.

To reduce the extent of buprenorphine radiolysis, a sterilization attempt at 15 kGy was performed (Figure S4). A lowering of the irradiation dose reduces the extent of degradation of pharmaceutical compounds [33]. After irradiation at 15 kGy, the pellets of microspheres loaded with buprenorphine were uncolored suggesting less advanced drug degradation than at 25 kGy. However, RP-HPLC analyses showed that degradation products were found in all PBS fractions collected during the first week of release, but in different proportions than during sterilization at 25 kGy, indicating that 15 kGy e-beam irradiation did not completely suppress buprenorphine radiolysis (Figure S4). A lower dose of irradiation altered the degradation dynamics of buprenorphine, but did not eliminate it, as the majority of the degradation products formed at 15 kGy were later released from DMic-2.

These experiments demonstrate the vulnerability of
buprenorphine to radiolysis by e-beam. Degradation of
buprenorphine after X-ray sterilization at 30 kGy was
already described after encapsulation in PLGA-based

<figure><img src="image_12.png" /><figcaption>**Fig. 4** RP-HPLC of polymyxin B eluted from DMic-3 after terminal sterilization at 15 kGy (A) and after extemporaneous loading on dry and sterile microspheres (B). Polymyxin B fractions were collected after 10 min (c), 48 h (e), 72 h (f) and 96 h (g) of release in PBS. Polymyxin B standard (50 µg/mL) (a) and PBS fraction of sterile drug-free DMic at 10 min (b). Detection was done at 205 nm</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**Fig. 5** RP-HPLC of buprenorphine eluted from DMic-2 after terminal sterilization at 25 kGy (A) and after extemporaneous loading on dry and sterile microspheres (B). Buprenorphine fractions were collected after 10 min (c), 5 h (d) and 48 h (e) of release in PBS. Buprenorphine standard (5 µg/mL) (a) and PBS fraction of sterile drug-free DMic-2 at 10 min (b). Detection was done at 280 nm. \*degradation products of buprenorphine</figcaption></figure>

microspheres, where approximately 25% of the buprenorphine content was degraded [11]. Our study confirms the radiosensitivity of buprenorphine to both X-ray and electron beam sterilization. The radiostability of opioids has been compiled and no evidence of major degradation has been described for morphine hydrochloride, codeine and methadone in their solid state [12]. For morphine hydrochloride, 1% to 5% of decomposition at 25 kGy was observed in two studies and no decomposition at 60 kGy in another study. These earlier radiostability studies were performed with ancient analytical tools such as TLC, which are less sensitive than current analytical instruments. The radiostability of opioids needs to be re-evaluated in the light of the present study and the recent work of Schreiner *et al.* (2021) [11].

To overcome the radiolysis barrier, buprenorphine was loaded on pre-sterilized DMic-2 through rapid mixing for 5 min (Figure S3 A). Subsequent immersion in PBS induced a sustained release of buprenorphine during degradation of the microspheres (Figure S3 C). Sustained release of buprenorphine was not affected by short loading time on sterilized microspheres. Chromatograms obtained for the release fractions collected after the extemporaneous loading showed the buprenorphine peak (retention time = 4.3 min) and a peak derived from DMic-2 (retention time = 5.05 min), but no secondary peaks derived from buprenorphine

(Fig. 5B). By rapidly loading buprenorphine onto pre-sterilized microspheres, as done in chemoembolization, the DrugMic platform enables sustained release of buprenorphine without the drawbacks of terminal sterilization causing buprenorphine radiolysis.

**Travoprost:** sterilization at 25 kGy of the travoprost loaded microspheres resulted in clear microsphere pellets. RP-HPLC analysis indicated that sterilization at this dose resulted in partial degradation of travoprost, highlighted by the detection of five secondary peaks corresponding to radiolysis products (Fig. 7). Travoprost degradation products generated during e-beam sterilization were released more quickly from DMic-2 than travoprost which was released gradually over 2 weeks (Fig. 3), then their levels decreased over time (Fig. 8A). The radiolytic by-products were more hydrophilic than travoprost, but closely related to travoprost, as indicated by the strong correlation with travoprost UV spectra (Fig. 8B). Detection at 280 nm suggested the formation of hydrophilic travoprost derivatives containing a phenoxy group. E-beam sterilization of travoprost-loaded DMic-2 generated radiolysis by-products; a similar instability of a prostamide compound (bimatoprost) used for glaucoma treatment has already been observed during gamma sterilization (25–35 kGy) of bimatoprost-loaded PLGA microspheres, where a 10% reduction in bimatoprost content was measured [49]. According to the results of this study and
<figure><img src="image_14.png" /><figcaption>**Fig. 6** Quantification of buprenorphine radiolysis products released from DMic-2 at each sampling time after sterilization at 25 kGy. The fractions recovered at each time during the buprenorphine release from DMic-2 were analyzed by RP-HPLC. The area of the 5 peaks of the degradation products produced after sterilization was related to the surface area of the buprenorphine peak (A). Spectra correlation between the degradation products and buprenorphine (B). 4 chromatograms were analysed at each time point</figcaption></figure>

<table><thead><tr><th>Peaks</th><th>Retention time (minutes)</th><th>Correlation with UV spectra of buprenorphine</th></tr></thead><tbody><tr><td>1</td><td>7.68</td><td>0</td></tr><tr><td>2</td><td>7.92</td><td>0.75</td></tr><tr><td>3</td><td>8.137</td><td>0</td></tr><tr><td>4</td><td>9.64</td><td>0.96</td></tr><tr><td>5</td><td>10.32</td><td>0.97</td></tr><tr><td>Buprenorphine</td><td>8.57</td><td>1</td></tr></tbody></table>

those of Hughes *et al.* (2006), special care must be taken when preparing long-acting glaucoma medications because of the radiosensitivity of prostaglandin analog during terminal sterilization.

## Active Substances without Evidence of Radiolysis

RP-HPLC analyses of fractions collected upon release in PBS did not show additional peaks for niflumic acid (retention time = 5.28 min) (Figure S5), tadalafil (retention time = 10.38 min) (Figure S6) and the teicoplanin massif (Figure S7), where teicoplanin was present as a major peak eluted at 7.45 min, which probably corresponds to the principal teicoplanin isoform, teicoplanin A2-2, representing 60% of the six components of teicoplanin [50]. For each active substance released from Drug-Mic after sterilization, the wavelength scans performed between 200 and 280 nm did not reveal the presence of any new peak (data not shown). According to RP-HPLC fractionation and DAD analysis (Table I), the 3 active substances loaded on DrugMic were not altered during e-beam sterilization. The stability of niflumic acid is

consistent with previous data reporting stability of other NSAIDs at solid state (ibuprofen, diclofenac sodium, naproxen, ketoprofen) after irradiation [13]. NSAIDs are resistant to radiolysis in the solid state after exposure to gamma and beta radiation, and this property makes them good candidates for the preparation of DDS sterilizable by irradiation. With regard to the radiostability of tadalafil, to the best of our knowledge, the effect of gamma or beta radiation on tadalafil in the dry state has not yet been described. These preliminary radiostability study, along with the sustained release data from DMic-2 (Fig. 3), suggest that tadalafil is a promising candidate for developing injectable sustained release formulations to improve treatment of erectile dysfunction, benign prostatic hyperplasia symptoms or pulmonary arterial hypertension since dysregulation of nitric oxide-cGMP-protein kinase G signaling plays a critical role in a variety of diseases [14, 39]. The absence of obvious signs of radiolysis of teicoplanin is in agreement with previous data showing the stability of vancomycin after gamma sterilization at various doses [12, 13]. A vancomycin derivative such as teicoplanin appeared equally resistant to degradation after e-beam
<figure><img src="image_15.png" /><figcaption>**Fig. 7** RP-HPLC of travoprost eluted from DMic-2 after final sterilization at 25 kGy. Fractions were collected after 10 min (c), 2 h (d) and 72 h (f) of release in PBS. Travoprost standard (2.5 µg/mL) (a) and PBS fractions of sterile drug-free DMic-2 at 10 min (b) and 72 h (e). Detection was done at 280 nm</figcaption></figure>

sterilization. Glycopeptides appear to be good candidates for the preparation of degradable and sterilisable DDS; their local administration in combination with systemic therapy would provide a high concentration of antibiotics that would be useful in the treatment of periprosthetic joint infections [16, 17].

## Radiolysis Sensitivity

Polymyxin B, buprenorphine and travoprost were particularly vulnerable to e-beam radiation even at low intensity. In peptides, the main products of irradiation are oxidation/hydroxylation (CH₂ → CH-OH), carbonylation (CH₂ → C=O) and peroxidation (CH₂ → CH₂-O-OH) following hydrogen abstraction at carbon α of amino acids [51]. Presumably, these modifications could be generated on threonine, phenylalanine and leucine residues constituting polymyxin B according to Minkoff's study of tripeptides exposed to electron radiation. In addition, di-aminobutyric acid residues of polymyxin B, structurally close to lysine, could be subject to oxidation/hydroxylation and peroxidation reactions. To explain the degradation of buprenorphine, its quaternary amine may be suspected, since quaternary amines of drugs (e.g. carfilzomib, atropine, tamoxifen)

are potential sites of cleavage by hydrated electrons [52] while Sun *et al.* [53] identify cleavage of the C-N bond of diclofenac upon exposure to an electron beam in water. This sensitivity of nitrogen atoms to radicals generated by radiation could explain why buprenorphine degrades during sterilization of drug loaded microspheres. For travoprost, the hydroxyl and fluoride groups as well as the double bonds and the ester group may be sensitive to degradation by free radicals [54, 55]. On contrary for niflumic acid and tadalafil, electrons are delocalized and therefore probably less accessible to free radicals. These structural differences may be the reason why some molecules are more susceptible to radiolysis than others. After e-beam sterilization of drug loaded DrugMic, the degradation products of buprenorphine, travoprost and polymyxin B exceeded the impurity thresholds specified in the ICH Q3 A guidelines, with the threshold varying depending on the maximum daily dose of the drug substance. For a drug with a maximum daily dose of ≤2 g/day, which is assumed for the molecules analyzed in this study, the threshold is typically 0.10%. For buprenorphine and travoprost, more than 0.1% of the degradation products were measured at each sampling time point, and for polymyxin B, the entire chromatographic massif was altered by e-beam irradiation, suggesting that most of the peptides were affected. Such degradation levels would require structural and toxicological studies, hence the interest in extemporaneous loading on DrugMic.

## Conclusion

This study demonstrates that various active substances can be efficiently loaded onto already prepared hydrophilic degradable microspheres through simple mixing, representing a break from PLGA-based drug delivery microspheres, where drugs are incorporated during synthesis [9, 11, 20, 40]. This strategy avoids exposure to organic solvents, high temperatures, and protect drugs them from potential degradation during final sterilization [56, 57]. A degradable hydrogel consisting of PEG formulated into microspheres accelerates the degradation of the DDS after drug release, with the aim to prevent the polymer from remaining in the tissue too long and causing injection site reactions. For example, the duration of buprenorphine release from DrugMic correlates with the degradation time of the microspheres (≈2 weeks), which is different from hydrophobic matrices composed of PLGA, lactide, ε-caprolactone, or their copolymers, where there is a gap between the release of buprenorphine over several days and the degradation of the material over months or years by hydrolysis or immune cell degradation [58, 59]. Because of the rapid degradation of DrugMic's PEG hydrogel, the risk of a long-term inflammatory reaction around the implant
<figure><img src="image_16.png" /><figcaption>**Fig. 8** Quantification of travoprost radiolysis products released from DMic-2 at each sampling time after sterilization at 25 kGy. The fractions recovered at each time during the release of travoprost loaded on DMic-2 were analyzed by RP-HPLC. The areas of the 5 peaks of degradation products generated during sterilization were related to the area of the travoprost peak (A). Spectra correlation between the degradation products and travoprost (B). 4 chromatograms were analysed at each time point</figcaption></figure>

B

<table><thead><tr><td>Peaks</td><td>Retention time (minutes)</td><td>Correlation with UV spectra of travoprost</td></tr></thead><tbody><tr><td>1</td><td>1.65</td><td>0.96</td></tr><tr><td>2</td><td>1.94</td><td>0.99</td></tr><tr><td>3</td><td>2.16</td><td>0.99</td></tr><tr><td>4</td><td>2.29</td><td>0.91</td></tr><tr><td>5</td><td>3.85</td><td>0.96</td></tr><tr><td>Travoprost</td><td>6.09</td><td>1</td></tr></tbody></table>

after drug delivery could be reduced. Compared to the DEBs [34, 35, 60, 61], the DrugMic approach expands the clinical indications beyond the treatment of liver cancer using local delivery of anticancer drugs (doxorubicin and irinotecan) to include acute pain management (niflumic acid, buprenorphine), anti-infective therapy (teicoplanin, polymyxin B) or ocular disease (travoprost).

The method of drug loading described in this study demonstrates that in the event of radiolysis of a pre-loaded active ingredient, it is still possible to achieve a sustained drug release without the formation of degradation products through the loading of pre-sterilized microspheres. In clinical settings the implications of the *in vivo* studies of the DrugMic platform could be the management of: (1) acute postoperative pain based on buprenorphine delivery for several days after surgery [18, 19], (2) bone and joint infections (BJI) by local delivery of polymyxin B and/or teicoplanin to target both gram-positive and gram-negative bacteria before degradation of the platform avoiding surgical removal as done with polymethylmethacrylate beads [16, 17, 62], (3) pelvic pain after embolization of uterine fibroid by local delivery of niflumic acid to reduce inflammation

induced by fibroid ischemia and necrosis during the first days after embolization (UFE) [25]; (4) glaucoma based on subconjunctival administration of degradable sustained release formulation of travoprost, the DrugMic degradation time should be extended to achieve several months of reliable delivery [63]. In practice, as with hospital-based chemoembolization for liver cancer using DEBs [34, 35, 60, 61], an extemporaneous drug loading step on the DrugMic would occur just prior to patient administration. Briefly, extemporaneous drug loading on DrugMic is easy to achieve and is performed in the same way as with non-degradable DEBs (DC beads® & DC beads LUMI® (Boston Scientific), Hepasphere® (Merit Medical), Lifepearl® (Terumo), which are mixed with aqueous solutions of doxorubicin, irinotecan or idarubicin at room temperature for about 1 h prior to patient administration [34, 35]. Furthermore, DrugMic can be manufactured using microfluidics (data not shown) to enable round-the-clock production of microspheres, making this approach to drug delivery scalable. Thus, crosslinked PEG-based microspheres can be used as degradable drug delivery platform adapted to the susceptibility of the active substances to radiolysis.
This study has limitations: firstly, it relies exclusively on *in vitro* data (loading, drug release, detection of degradation products by HPLC), concerned few active substances (n = 6) and did not provide *in vivo* data such as pharmacokinetics blood profiles upon subcutaneous injection of drug loaded microspheres. Detection of radiolysis was only based by HPLC and has not been supported by other methods, such electron spin resonance (ESR) spectroscopy to detect free radicals in the sterilized samples or *in vitro* evaluation of antibiotic activity after sterilization. The researchers' affiliation and co-inventorship of related patents could have introduced a bias. Therefore, it would be beneficial to have independent validation to further strengthen the findings.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1208/s12249-025-03144-1.

**Authors Contributions** LB, AB, LM: writing—original draft; AB, ES, LB: doing all *in vitro* studies; LM: conceptualization, LB, AB, LM: review & editing; LM: supervision and final approval of the version to be published.

**Funding** Open access funding provided by Université Paris-Saclay.

**Data Availability** Data will be made available on request.

## Declarations

**Competing Interest** The authors (LB, AB, ES, LM) are co-inventors of patents on resorbable microspheres co-owned by Occlugel company and Public Institutions (CNRS, AP-HP, Universities of Paris VII & XI). AB, ES, LB are employees of Occlugel.

**Disclosures** The research being reported in this publication was funded by Occlugel Company. The authors (LB, AB, ES, LM) are co-inventors of patents on resorbable microspheres co-owned by Occlugel company and Public Institutions (CNRS, AP-HP, Universities of Paris VII & XI).

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

## References

1. Abuhanoğlu G, Özer AY. Radiation sterilization of new drug delivery systems. Interv Med Appl Sci. 2014;6:51-60. https://doi.org/10.1556/imas.6.2014.2.1.

2. Domańska IM, Oledzka E, Sobczak M. Sterilization process of polyester based anticancer-drug delivery systems. Int J Pharm. 2020;587:119663. https://doi.org/10.1016/j.ijpharm.2020.119663.

3. Lambert BJ, Hansen JM, Bryans TD, Lam S. Sterility Assurance Across-Sectors—New Paradigms and Tools. Front Med Technol 2021;3. https://doi.org/10.3389/fmedt.2021.622710.

4. Filipczak K, Wozniak M, Ulanski P, Olah L, Przybytniak G, Olkowski RM, Lewandowska-Szumiel M, Rosiak JM. Poly(ε-caprolactone) Biomaterial Sterilized by E-Beam Irradiation. Macromol Biosci. 2006;6:261-73. https://doi.org/10.1002/mabi.200500215.

5. Spenlehauer G, Vert M, Benoit J, Boddaert A. In vitro and In vivo degradation of poly(D, L lactide/glycolide) type microspheres made by solvent evaporation method. Biomaterials. 1989;10:557-63. https://doi.org/10.1016/0142-9612(89)90063-X.

6. Montanari L, Cilurzo F, Conti B, Genta I, Groppo A, Valvo L, Faucitano A, Buttafava A. Gamma irradiation effects and EPR investigation on poly(lactide-co-glycolide) microspheres containing bupivacaine. Farm. 2002;57:427-33. https://doi.org/10.1016/S0014-827X(02)01220-X.

7. Faisant N, Siepmann J, Oury P, Laffineur V, Bruna E, Haffner J, Benoit J. The effect of gamma-irradiation on drug release from bioerodable microparticles: a quantitative treatment. Int J Pharm. 2002;242:281-4. https://doi.org/10.1016/S0378-5173(02)00188-6.

8. Lee JS, Chae GS, Khang G, Kim MS, Cho SH, Lee HB. The effect of gamma irradiation on PLGA and release behavior of BCNU from PLGA wafer. Macromol Res. 2003;11:352-6. https://doi.org/10.1007/BF03218376.

9. Fernández-Carballido A, Herrero-Vanrell R, Molina-Martínez IT, Pastoriza P. Sterilized ibuprofen-loaded poly(D, L-lactide-co-glycolide) microspheres for intra-articular administration: effect of γ-irradiation and storage. J Microencapsul. 2004;21:653-65. https://doi.org/10.1080/09687860400008437.

10. de Oliveira AR, Mesquita PC, Machado PRL, Farias KJS, de Almeida YMB, Fernandes-Pedrosa MF, Cornélio AM, do Egito EST, da Silva-Júnior AA. Monitoring structural features, bio-compatibility and biological efficacy of gamma-irradiated methotrexate-loaded spray-dried microparticles. Mater Sci Eng C. 2017;80:438-48. https://doi.org/10.1016/j.msec.2017.06.013.

11. Schreiner V, Detampel P, Jirkof P, Puchkov M, Huwyler J. Buprenorphine loaded PLGA microparticles: characterization of a sustained-release formulation. J Drug Deliv Sci Technol. 2021;63:102558. https://doi.org/10.1016/j.jddst.2021.102558.

12. Boess C, Bögl KW. Influence of radiation treatment on pharmaceuticals-a review: alkaloids, morphine derivatives, and antibiotics. Drug Dev Ind Pharm. 1996;22:495-529. https://doi.org/10.3109/03639049609108354.

13. Jacobs GP. Irradiation of pharmaceuticals: a literature review. Radiat Phys Chem. 2022;190:109795. https://doi.org/10.1016/j.radphyschem.2021.109795.

14. von Jehle DK, Sunesra R, Uddin H, Paul KK, Joglar AA, Michler OD, Blackwell TA, Gaalema D, Hayek S, Jneid H. Benefits of tadalafil and sildenafil on mortality, cardiovascular disease, and dementia. Am J Med 0 2024. https://doi.org/10.1016/j.amjmed.2024.10.039.

15. Baryakova TH, Pogostin BH, Langer R, McHugh KJ. Overcoming barriers to patient adherence: the case for developing innovative drug delivery systems. Nat Rev Drug Discov. 2023;22(5):387-409. https://doi.org/10.1038/s41573-023-00670-0.

16. ter Boo G-JA, Grijpma DW, Moriarty TF, Richards RG, Eglin D. Antimicrobial delivery systems for local infection prophylaxis in orthopedic- and trauma surgery. Biomaterials. 2015;52:113-25. https://doi.org/10.1016/j.biomaterials.2015.02.020.

17. Meeker DG, Cooper KB, Renard RL, Mears SC, Smeltzer MS, Barnes CL. Comparative study of antibiotic elution profiles from alternative formulations of polymethylmethacrylate bone cement.
J Arthroplasty. 2019;34:1458–61. https://doi.org/10.1016/j.arth. 2019.03.008.

18. Lee S-O, Huang L-P, Wong C-S. Preoperative administration of extended-release dinalbuphine sebacate compares with morphine for post-laparoscopic cholecystectomy pain management: a randomized study. J Pain Res. 2020;13:2247–53. https://doi.org/10. 2147/JPR.S263315.

19. Zheng Z-H, Yeh TT, Yeh CC, Lin PA, Wong CS, Lee PY, Lu CH. Multimodal analgesia with extended-release dinalbuphine sebacate for perioperative pain management in upper extremity trauma surgery: a retrospective comparative study. Pain Ther. 2022;11:643–53. https://doi.org/10.1007/s40122-022-00383-z.

20. Fedorchak MV, Conner IP, Medina CA, Wingard JB, Schuman JS, Little SR. 28-day intraocular pressure reduction with a single dose of brimonidine tartrate-loaded microspheres. Exp Eye Res. 2014;125:210–6. https://doi.org/10.1016/j.exer.2014.06.013.

21. Natarajan JV, Darwitan A, Barathi VA, Ang M, Htoon HM, Boey F, Tam KC, Wong TT, Venkatraman SS. Sustained drug release in nanomedicine: a long-acting nanocarrier-based formulation for glaucoma. ACS Nano. 2014;28:419–29. https://doi.org/10.1021/ nn4046024.

22. Kapil V, Louthan J, Hamedi N, Antoniou S, Lobo M. [PP.22.06] Transdermal clonidine in difficult-to-treat hypertension patients attending a specialist BP clinic in the United Kingdom. J Hyper tens. 2017;35:e279. https://doi.org/10.1097/01.HJH.0000523811. 90036.AC.

23. Louguet S, Verret V, Bédouet L, Servais E, Pascale F, Wassef M, Labarre D, Laurent A, Moine L. Poly(ethylene glycol) meth acrylate hydrolyzable microspheres for transient vascular embo lization. Acta Biomater. 2014;10:1194–205. https://doi.org/10. 1016/j.actbio.2013.11.028.

24. Maeda N, Verret V, Moine L, Bédouet L, Louguet S, Servais E, Osuga K, Tomiyama N, Wassef M, Laurent A. Targeting and recanalization after embolization with calibrated resorbable microspheres versus hand-cut gelatin sponge particles in a porcine kidney model. J Vasc Interv Radiol. 2013;24:1391–8. https://doi.org/ 10.1016/j.jvir.2013.05.058.

25. Verret V, Pelage JP, Wassef M, Louguet S, Servais E, Bédouet L, Beaulieu T, Moine L, Laurent A. A novel resorbable embolization microsphere for transient uterine artery occlusion: a comparative study with trisacryl-gelatin microspheres in the sheep model. J Vasc Interv Radiol. 2014;25(2014):1759–66. https://doi.org/10. 1016/j.jvir.2014.06.025.

26. Bédouet L, Verret V, Louguet S, Servais E, Moine L, Laurent A. Doxorubicin, irinotecan and sunitinib: loading and release with a resorbable embolization microsphere (REM). J Vasc Interv Radiol. 2013;24:S48. https://doi.org/10.1016/j.jvir.2013. 01.109.

27. Bédouet L, Verret V, Louguet S, Servais E, Pascale F, Beilvert A, Baylatry MT, Labarre D, Moine L, Laurent A. Anti-angiogenic drug delivery from hydrophilic resorbable embolization microspheres: An in vitro study with sunitinib and bevacizumab. Int J Pharm. 2015;484:218–27.

28. Bushell JA, Claybourn M, Williams HE, Murphy DM. An EPR and ENDOR study of γ- and β-radiation sterilization in poly (lactide-co-glycolide) polymers and microspheres. J Control Release. 2005;110:49–57. https://doi.org/10.1016/j.jconrel.2005.09.009.

29. Magnon L, Laurent A, Wassef M, Bédouet L, Louguet S, Verret V, Servais E. Implantable bio-resorbable polymer charged with fragile macromolecules. WO2012120139A1, 2012.

30. Magnon L, Laurent A, Wassef M, Bedouet L, Louguet S. Verret V, Servais E. Implantable swellable bio-resorbable polymer. WO2012120138A1, 2012.

31. Caine M, Zhang X, Hill M, Guo W, Ashrafi K, Bascal Z, Kilpat- rick H, Dunn A, Grey D, Bushby R, Bushby A, Willis SL, Dreher MR, Lewis AL. Comparison of microsphere penetration with LC

Bead LUMI™ versus other commercial microspheres. J Mech Behav Biomed Mater. 2018;78:46–55. https://doi.org/10.1016/j. jmbbm.2017.10.034.

32. Maquille A, Jiwan J-LH, Tilquin B. Radiosterilization of drugs in aqueous solutions may be achieved by the use of radioprotective excipients. Int J Pharm. 2008;349:74–82. https://doi.org/10. 1016/j.ijpharm.2007.07.024.

33. Slegers C, Tilquin B. Final product analysis in the e-beam and gamma radiolysis of aqueous solutions of metoprolol tartrate. Radiat Phys Chem. 2006;75:1006–17. https://doi.org/10.1016/j. radphyschem.2005.12.020.

34. Jordan O, Denys A, De Baere T, Boulens N, Doelker E. Com- parative study of chemoembolization loadable beads: In vitro drug release and physical properties of DC bead and hepasphere loaded with doxorubicin and irinotecan. J Vasc Interv Radiol. 2010;21:1084–90. https://doi.org/10.1016/j.JVIR.2010.02.042.

35. de Baere T, Plotkin S, Yu R, Sutter A, Wu Y, Cruise GM. An In vitro Evaluation of Four Types of Drug-Eluting Microspheres Loaded with Doxorubicin. J Vasc Interv Radiol. 2016;27:1425– 31. https://doi.org/10.1016/j.JVIR.2016.05.015.

36. Bédouet L, Moine L, Servais E, Beilvert A, Labarre D, Laurent A. Tunable delivery of niflumic acid from resorbable embolization microspheres for uterine fibroid embolization. Int J Pharm. 2016;511:253–61. https://doi.org/10.1016/j.ijpharm.2016.06. 128.

37. Wassef M, Pelage JP, Velzenberger E, Namur J, Schwartz-Cornil I, Taylor RR, Lewis AL, Laurent A. Anti-inflammatory effect of ibuprofen-loaded embolization beads in sheep uterus. J Biomed Mater Res B Appl Biomater. 2008;86:63–73. https://doi.org/10. 1002/jbm.b.30988.

38. Verret V, Bevilacqua C, Schwartz-Cornil I, Pelage JP, Wassef M, Namur J, Bédouet L, Lewis AL, Martin P, Laurent A. IL6 and TNF expression in vessels and surrounding tissues after embolization with ibuprofen-loaded beads confirms diffusion of ibuprofen. Eur J Pharm Sci. 2011;42:489–95. https://doi.org/10.1016/j.ejps. 2011.02.001.

39. Samidurai A, Xi L, Das A, Kukreja RC. Beyond erectile dysfunc- tion: cGMP-specific phosphodiesterase 5 inhibitors for other clini- cal disorders. Annu Rev Pharmacol Toxicol. 2023;63:585–615. https://doi.org/10.1146/annurev-pharmtox-040122-034745.

40. Yenİce İ, Çaliş S, Atılla B, Kaş HS, ÖM, Ekİzoğlu M, Bİlgİll HAA. Hincal, In vitro/in vivo evaluation of the efficiency of teicoplanin-loaded biodegradable microparticles formulated for implantation to infected bone defects. J Microencapsul. 2003;20:705–717. https://doi.org/10.1080/026520403100015 4179.

41. Cai Q, Bei J, Wang S. Synthesis and degradation of a tri- component copolymer derived from glycolide, L-lactide, and ε-caprolactone. J Biomater Sci Polym. 2000;11:273–88. https:// doi.org/10.1163/156856200743698.

42. Qian H, Bei J, Wang S. Synthesis, characterization and degrada- tion of ABA block copolymer of l-lactide and ε-caprolactone. Polym Degrad Stab. 2000;68:423–9. https://doi.org/10.1016/ S0141-3910(00)00031-8.

43. Netti PA, Biondi M, Frigione M. Experimental studies and mod- eling of the degradation process of Poly(Lactic-co-Glycolic Acid) microspheres for sustained protein release. Polym. 2020;12:2042. https://doi.org/10.3390/POLYM12092042.

44. Lim LW, Tan WS, Ho KL, Mariatulqabtiah AR, Abu Kasim NH, Abd Rahman N, Wong TW, Chee CF. Challenges and compli- cations of Poly(lactic-co-glycolic acid)-based long-acting drug product development. Pharmaceutics. 2022;14:614. https://doi. org/10.3390/pharmaceutics14030614.

45. Orwa JA, Van Gerven A, Roets E, Hoogmartens J. Liquid chromatography of polymyxin B sulphate. J Chromatogr A.
2000;870:237-43. https://doi.org/10.1016/S0021-9673(99)00936-X.

46. Kassamali Z, Prince RA, Danziger LH, Rotschafer JC, Rhomb-erg PR, Jones RN. Microbiological Assessment of Polymyxin B Components Tested Alone and in Combination. Antimicrob Agents Chemother. 2015;59:7823-5. https://doi.org/10.1128/AAC.01021-15.

47. Fleurette J, Madier S, Transy MJ. Bacteriostatic activity of vari-ous antibiotics after gamma-ray irradiation. Int At Energy Agency IAEA 1975;539.

48. Horne T. Biochemical applications of large radiation sources with special reference to pharmaceutical products, in: Proc. 2nd United Nations Int Conf Peac Uses At Energy, Held Geneva, 1 Sept. 13 Sept. 1958., UN, 1958. pp. 276-342.

49. Hughes P, Chan-Lin J-E, Welty DF. US20060182781A1 - Meth-ods for treating ocular conditions with cyclic lipid contraining microparticles. 2006. https://patents.google.com/patent/US20060182781A1/en?oq=US-2006182781-A1+(2006). Accessed 10 Jan 2025.

50. Lee J, Chung EK, Kang SW, Lee HJ, Rhie SJ. Quantification of teicoplanin using the HPLC-UV method for clinical applications in critically ill patients in korea. Pharmaceutics. 2021;13:572. https://doi.org/10.3390/pharmaceutics13040572.

51. Minkoff BB, Bruckbauer ST, Sabat G, Cox MM, Sussman MR. Covalent modification of amino acids and peptides induced by ionizing radiation from an electron beam linear accelerator used in radiotherapy. Radiat Res. 2019;191:447-59. https://doi.org/10.1667/RR15288.1.

52. Guo Z, Hong H, Zheng Y, Wang Z, Ding Z, Fu Q, Liu Z. Radiotherapy-induced cleavage of quaternary ammonium groups activates prodrugs in tumors. Angew Chem Int Ed Engl. 2022;61:e202205014. https://doi.org/10.1002/anie.202205014.

53. Sun Y, Madureira J, Justino GC, Cabo Verde S, Chmielewska-Śmietanko D, Sudlitz M, Bulka S, Chajduk E, Mróz A, Wang S, et al. Diclofenac degradation in aqueous solution using electron beam irradiation and combined with nanobubbling. Appl Sci. 2024;14:6028. https://doi.org/10.3390/app14146028.

54. Vlasov VM. Fluoride ion as a nucleophile and a leaving group in aromatic nucleophilic substitution reactions. J Fluor Chem. 1993;61:193-216. https://doi.org/10.1016/S0022-1139(00)80104-9.

55. L. González-López, L. T. Kearney, C. Janke, J.F. Wishart, A.K. Naskar, N. Kanbargi, F.B. Bateman, M. Al-Sheikhly, On the

electron beam-induced degradation of vinyl ester thermosets. Polym Degradation Stab. 2023;211:110307. https://doi.org/10.1016/j.polymdegradstab.2023.110307.

56. Ibrahim MA, Ismail A, Fetouh MI, Göpferich A. Stability of insulin during the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres. J Control Release. 2005;106:241-52. https://doi.org/10.1016/J.JCONREL.2005.02.025.

57. Houchin ML, Topp EM. Chemical degradation of peptides and proteins in PLGA: a review of reactions and mechanisms. J Pharm Sci. 2008;97:2395-404. https://doi.org/10.1002/JPS.21176.

58. Meek MF, Jansen K. Two years after *in vivo* implantation of poly(-DL-lactide-ε-caprolactone) nerve guides: Has the material finally resorbed? J Biomed Mater Res Part A. 2009;89A:734-8. https://doi.org/10.1002/jbm.a.32024.

59. Haertel AJ, Schultz MA, Colgin LM, Johnson AL. Predictors of subcutaneous injection site reactions to sustained-release buprenorphine in rhesus macaques (*Macaca mulatta*). J Am Assoc Lab Anim Sci. 2021;60:329-36. https://doi.org/10.30802/AALAS-JAALAS-20-000118.

60. Fuchs K, Duran R, Denys A, Bize PE, Borchard G, Jordan O. Drug-eluting embolic microspheres for local drug delivery – State of the art. J Control Release. 2017;262:127-38. https://doi.org/10.1016/j.jconrel.2017.07.016.

61. Nam HC, Jang B, Song MJ. Transarterial chemoembolization with drug-eluting beads in hepatocellular carcinoma. World J Gastro-enterol. 2016;22:8853-61. https://doi.org/10.3748/wjg.v22.i40.8853.

62. Steadman W, Chapman PR, Schuetz M, Schmutz B, Trampuz A, Tetsworth K. Local antibiotic delivery options in prosthetic joint infection. Antibiotics. 2023;12:752. https://doi.org/10.3390/antibiotics12040752.

63. Chong RS, Su DH, Tsai A, Jiang Y, Htoon HM, Lamoureux EL, Aung T, Wong TT. Patient acceptance and attitude toward an alternative method of subconjunctival injection for the medical treatment of glaucoma. J Glaucoma. 2013;22:190-4. https://doi.org/10.1097/IJG.0b013e318237c6c4.

**Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.