43 Remotely controllable microrobots are appealing for various biomedical in vivo applications. 44 In particular, our group has focused in the last years in developing sperm -microcarriers to assist 45 sperm cells with motion deficiencies or low count (two of the most prominent male infertility 46 problems) to reach the oocyte towards in vivo assisted fertilization . Different sperm carriers 47 considering their motion in realistic media and confined environments have been optimized . 48 However, the already reported s perm carriers have been mainly designed to transport single 49 sperm cells , with limited functionality. Thus, to take a step forward, here we propose the 50 development of a 4D-printed multifunctional microcarrier containing soft and smart materials, 51 which can not only transport and deliver multiple sperm cells, but also release heparin and 52 mediate local enzymatic reactions by hyaluronidase -loaded polymersomes (HYAL -Psomes ). 53 These multifunctional facets enable in-situ (i) sperm capacitation/ hyperactivation, and (ii) local 54 degradation of the cumulus complex that surrounds the oocyte, both t o facilitate the sperm - 55 oocyte interaction for the ultimate goal of in vivo assisted fertil ization. 56 3 57 1. Introduction 58 A new generation of remote controllable soft and smart microrobots, propelled/guided by 59 endogenous (e.g., chemotaxis), or exogenous (e.g., ultrasound, magnetic field, electric field) 60 stimuli, has expanded the field of biomedical sciences by creating unique opportunities for less - 61 invasive medical operations in vivo . Microrobots have already being use d in small animals, for 62 example , to treat stomach infection or for delivering anticancer drugs .[1–6] These microrobots 63 are characterized by their propulsion mech anism, which may be chemical, physical, or 64 biohybrid.[7] In particular, microrobots propelled by motile cells or microorganisms have been 65 widely used , as they take advantage of the taxis and cell -interaction abilities of the living 66 components with the cont rol/steerability and the imaging enhancement of the synthetic 67 microparts .[8] 68 Among different types of motile microorganisms and cells, bacteria and spermatozoa are 69 promising candidates for delivering therapeutic cargo es. This is due to their very efficient 70 propulsion mechanism, flagellum beating, which allows them to adapt to different 71 microenvironments and to swim in complex fluids —even against the flow.[9–11] Among 72 bacterial strains, Magnetococcus marinus “MC -1”, known as magnetotactic bacteria (MTB), 73 has been used for drug -delivery applications, as it can be genetically modified using tools of 74 synthetic biology to improve its targeting ability and to reduce its pathogenic nature.[12–16] 75 Additionally, the magnetosomes in the M. marinus cell not only offer the possibility of external 76 magnetic guidance, but also enable their tracking via magnetic resonance imaging (MRI).[2,17] 77 Spermatozoa (sperm cells) are other promising biological component s for constructing 78 biohybrid microrobots, as the y are inherently optimized to swim through complex fluids and 79 organs, such as those in the reproductive system, without producing harmful immunoreactions. 80 Besides their natural function of fertilization, sperm cells exhibit a high drug encapsulation 81 capabi lity along with a high drug carrying stability, which makes them suitable for various 82 gynecological healthcare applications.[18,19] Moreover, their chemotaxis ( tendency to swim 83 toward a higher concentration of extracellular signals ), thermotaxis (tendency to swim towards 84 a temperature gradient), thigmotaxis (tendency to swim near the surface) , and rheotaxis 85 (swimming against the fluid flow), abilities offer a wide range of options for their motion 86 control , making them promising medical vehicles for minimally -invasive operations.[20–26] 87 4 Our group was the first to employ sperm cell s as component s of microrobotics systems,[18] in 88 particular towards the development of novel in vivo assisted fertilization methods, where sperm 89 cells with motion deficiencie s (asthenospermia) or low count (oligospermia) could be assisted 90 by engineered microrobots.[27,28] In fact, the capture, guidance and release of single sperm cells 91 employing different synthetic microstructures have been demonstrated.[19] Optimizations on the 92 microstructures geometry have also been carried out, considering parameters such as sperm 93 morphology and tail beating amplitude , as well as, the temperature and viscosity of the 94 surrounding fluid.[29,30] Furthermore, the release of a single sperm cell has been demonstrated 95 using either thermo -responsive rolled -up microtubes, or tetrapod -like microstructures with 96 bending arms.[31,32] In the case of motion deficient sperm cells, micropropellers based on helical 97 structures were developed to perform cargo d elivery to the oocyte ,[27] or more recently sperm 98 were decorated with magnetic particles for their propulsion under oscillating magnetic 99 fields.[19,33] 100 The vision of this project is to assist sperm cells to reach the fertilization site in in vivo scenarios, 101 when the sperm count is low (<20 million sperm per mL), also called oligospermia. Current 102 treatments for this male infertility problem are based on in vitro fertilization (IVF) and 103 intracytoplasmic sperm injection (ICSI). The first one consists of incubating the preselected 104 sperm cells (millions of them) nearby the retrieved oocytes. On the other side, in ICSI just one 105 sperm is picked up and directly in jected into the oocyte cytoplasm. In both cases, the 106 fertilization rates are quite high, above 93.4% based on the German register for IVF 107 procedures.[34] However, the problem persists when looking at the embryo implantation rates, 108 which are 26.3% and 29.4% after the transfer of a 1 -day and 5/6 -day cultured embryo, 109 respectively. These low implantation rates might be due to the oxidative stress that gametes 110 experience during retrieval, washing, selection, and incubation steps, processes that are known 111 to affe ct gamete quality. Thereupon, we aimed to assist those sperm cells in vivo conditions to 112 perform the fertilization process under more natural conditions. To accomplish this goal, we 113 are developing different approaches to transport a high number of sperm ce lls as well as to 114 increase the functionality of the microcarrier. 115 Here , we employ both a non -stimuli -responsive polymer (IPS photoresist) and a thermo - 116 responsive hydrogel poly(N -isopropylacrylamide) (PNIPAM), for the fabrication of 4D printed 117 sperm microca rriers via two -photon polymerization (TPP) (Figure 1a) . In our recent review 118 article, the fundamentals of 3D and 4D lithography of untethered microrobots were 119 comprehensively reviewed.[35] Among the stimuli -responsive materials, hydrogel -based 120 5 polymers are versatile as they can undergo reversible solubility transition upon application of 121 certain stimuli (e.g., temperature, salt concentration, pH).[36] One of the most well -studied 122 stimuli -responsive hydrogels is PNIPAM, which responds to temperature change s. Its thermo - 123 responsiveness is based on a reversible phase transition from a swollen hydrophilic state at 124 temperatures below its lower critical solution temperature (LCST) to a collapsed hydrophobic 125 state at temperatures above its LCST.[37–41] Therefore, we hereby used PNIPAM to equip the 126 microcarriers with soft and smart microgates for triggering the release of heparin and motile 127 sperm cells , for future in vivo assisted fertilization. Moreover, we functionalized the 128 microcarriers with a specific nanocarrier platform to locally mediate the degradation of 129 hyaluronic acid (HA) found in the oocyte -cumulus complex.[42,43] Thus, b y taking advantage of 130 smart materials and novel microfabrication techniques, we present here a multifunctional 131 microcarrier able to: (i) transport and deliver multiple motile sperm cells, to increase the 132 chances of fertilization, (ii) in -situ capacitate /hyperactivate the sperm cells through the local 133 release of heparin, and (iii) assist the degradation of the HA -cumulus complex, by the local 134 action of HYAL -Psomes previously functionalized on the microcarrier’s surface. The 135 microcarriers are also coated with a metallic layer (Au) to enhance their visualization via dual 136 ultrasound (US)/photoaco ustic (PA) imaging, which makes them potential candidates for future 137 in vivo applications . 138 2. Results and Discussion 139 2.1. Concept and fabrication of the 4D -printed microcarriers 140 In this work, a modified protocol for the synthesis of PNIPAM was employed (see the 141 Experimental Section in the Supplementary information) .[44] The whole fabrication process 142 of the proposed microcarrier is depicted in Figure 1a , comprising the following steps: 143 fabrication of the supporting structure, patterning of the thermo -responsive mic rogate, coating 144 with metal -oxide for magnetic guidance and imaging contrast enhancement, and two final 145 functionalization steps to load heparin into the PNIPAM microgate and to immobilize HYAL - 146 Psomes onto the microcarrier’s surface. In Figure 1b ,i, the opti cal microscopy image shows 147 the microcarrier coupling with multiple motile sperm cells. In the fluorescence microscopy 148 image, both the non -stimuli -responsive (passive) and stimuli -responsive (active) components 149 of the microcarrier are distinguishable (Figur e 1b ,ii). The green color represents IPS 150 photoresist, a non -stimuli -responsive material which is autofluorescent, and the red color 151 represents PNIPAM, a thermo -responsive hydrogel which was mixed with Rhodamine B (RhB) 152 prior polymerization . 153 6 Bovine sperm cells were chosen due to their availability and similar morphology to human 154 sperm cells. All mammalian sperm cells consist of a head, a midpiece , and a flagellum. Bovine 155 sperm cells are about 60 –70 μm long including the sperm head which is approximately 10 μm 156 long and 5 μm in diameter. The head has an oval, flat shape and is approximately 1 μm in 157 thickness.[45] Therefore, the microcarrier was designed with multiple cavities , each of them with 158 an overall size of 8 × 8 µm2. The cavities can be observed in the scanning electron microscopy 159 (SEM) image (Figure 1b ,iii). This microfabrication design enables us to keep the captured 160 sperm cells inside the microcarrier during transport while propelling it via the sperm cells’ tail 161 beating . Furthermore, we considered p revious hydrodynamic studies on different sperm -hybrid 162 microrobot “spermbots”, in which the stream -lined cap design resulted in efficient spermbot 163 motion, even in ex vivo oviduct fluid and highly viscous medium.[30] The general concept of 164 the suggested multi -material microcarriers is shown in Figure 1c . In Figure 1d , i-ii, the 165 biocompatibility of all employed components of the microcarrier was assessed using a MTS - 166 Assay, confirming that there was no significant effect on Madin Darby Canine Kidney (MDCK) 167 cells’ viability ( details in the Supplementary information ). Regarding the pot ential 168 inflammatory response of the sperm micro carriers, a previous study by our group shows that 169 after incubating the microcarriers in ex vivo human whole blood ,[46] no coagulation and 170 granulocyte activation indicators were present in comparison with the bare control sample (only 171 blood), demonstrating that neither a specific immune response nor inflammation was induced 172 by sperm or the sperm microcarriers. This experime nt was done using Chandler loops (which 173 employs an ELISA assay), where the blood was incubated with sperm cells and microrobots for 174 2 h. Also, cytotoxicity assays employing sperm and various sperm carriers have been performed 175 in previously published works of our group, showing a non -evident cytotoxicity effect.[27,32] 176 7 177 Figure 1. a) Fabrication steps of the 4D -printed microcarrier, 1. TPP of IPS photoresist (base structure), 178 2. Drop casting of PNIPAM and alignment, 3. TPP of PNIPAM (microgate), 4. Coating T i/Fe/Au/SiO 2 179 onto the microcarriers for further magnetic guidance/imaging/surface functionalization, and two 180 functionalization steps: 5. Heparin loading into the polymeric networks of PNIPAM, and 6. 181 Immobilization of HYAL -Psomes on the microcarrier’s surfa ce, b) (i) Optical microscopy image of the 182 resulting microcarrier with captured multiple motile sperm cells, (ii) Fluorescence microscopy image 183 showing both the passive (green) and active (red) components of the microcarrier, and (iii) 184 Corresponding SEM im age highlighting the cavities where sperm cells will be coupled, c) Concept of 185 the multifunctional microcarrier with heparin (used for sperm hyperactivation) and hyaluronidase (used 186 for cumulus cell removal), and the expected natural stimuli (pH and temperature varying from the uterus 187 to the fertilization site) , d) Biocompatibility test, (i) MTS -assay results which show biocompatibility of 188 8 microcarrier components (n=5000, N=6, mean, ±SD), and (ii) SEM image of MDCK cells in contact 189 with microcarriers. 190 2.2. Characterization of the 4D -printed stream -lined microcarriers 191 As explained above, the proposed 4D -printed stream -lined microcarrier, consisting of a base 192 microstructure made of passive photoresist IPS (non -stimuli -responsive) and an adaptable 193 microgate m ade of active PNIPAM hydrogel (thermo -responsive), was fabricated via TTP. 194 The geometry of the microcarrier was designed by considering previously reported works of 195 our group, in which the performance improvement of th e stream -lined cap compared to the 196 cylindrical one was analyzed. [30,47] As expected, the tubular microrobot was more prone to 197 obstruct by the obstacles in the fluid, compared to the stream -lined cap which experienced lower 198 hydrodynamic pressures and a stable wake flow around it. This characteristic makes the stream - 199 lined cap to consume lower energy to swim forward which leads to less consumption of the 200 sperm’s energy . We summarized t he process of the geometrical optimization of microcarrier in 201 Figure S1 (detail information and results can be found in the Supplementary information) . 202 In order to optimize the response of the PNIPAM microgate, different parameters were 203 considered during t he fabrication of the structures, namely the laser power (LP) and the scan 204 speed (SS). These two parameters regulate the local exposure dose which affects the degree of 205 hydrogel cross -linking. 206 First, cubic microstructures with the size of 30 × 30 × 10 µm3 were fabricated using different 207 LP and SS. After polymerization, the sample was developed and tested by using a Peltier 208 element mounted underneath the sample to change the temperature of the solution in which the 209 structures were immersed (detail in Supple mentary information; Video S1) . It is known that 210 high cross -linked polymer results from a high exposure dose either by increasing the LP or the 211 time of exposure (decreasing the SS), leading to a lower temperature response (referred to as 212 hard cube).[44] Likewise, using lower LP or higher SS (lower exposure dose) results in a lower 213 cross -linked polymer with higher thermo -response (referred to as soft cube) (Figure 2a , i-ii). 214 These varying parameters allow us to exploit a variety of thermo -responsive properti es within 215 the same material (Figure 2a , ii). Figure 2a , iii shows the reversible shape change of the soft 216 cube with maximum response upon temperature change. 217 In this work, the PNIPAM microgate was fabricated in three dimensions (3D), so the effect of 218 varyi ng heights (corresponding to the volume) on its thermo -responsive properties was also 219 9 studied. Based on a previous study conducted by Hippler and colleagues , the thermal expansion 220 coefficients and Young’s modulus of PNIPAM depend significantly on the struc ture volume.[44] 221 To investigate this effect, different cubic microstructures with different heights were fabricated 222 (within the size range of the required microgate, see Figure 2b ). Figure 2b demonstrates that 223 when the height of the PNIPAM cubes increases, the thermo -response of the cubes also 224 increases from ca. 700 µm2 (in cubes with a height of 10 µm) to ca. 1400 µm2 (in cubes with a 225 height of 30 µm), almost doubling. Based on these results, we can conclude that the thermo - 226 response of PNIPAM is in all dim ensions, and the shape changes are also more pronounced 227 when the volume of the polymeric structure is higher. 228 The initial characterization of the 3D -printed PNIPAM microstructures was carried out in the 229 temperature range from 25°C to 40°C to observe the th ermal actuation of PNIPAM in the whole 230 range to identify the lower critical solution temperature (LCST) of the 4D printed 231 microstructures. Then, by knowing the thermo -responsive properties of PNIPAM at the 232 microscale, the design of the microgate for the release of multiple sperm cells was optimized . 233 The microgate has to guarantee the prevention of unwanted sperm release during transport and 234 provide enough space for releasing them at the physiological temperature (ca. 38°C, in the luteal 235 phase before fertilization).[48] To accomplish this goal, different designs for the microgate were 236 investigated ( Figure 2c , i-iii; Video S2) . After several design optimizations, and based on the 237 size of the sperm head (10 × 5 µm2), design number ( Figure 2c, iii) was the most promising, 238 exhibiting major shape change, thus ensuring enough space for the sperm release. In Figure 2c , 239 iv the microcarrier components in the microgate area are shown under fluorescence microscopy, 240 where we can distinguish the active part (PNI PAM) from the passive part (IPS) with different 241 colors, red and green, respectively. Here the height of the PNIPAM was ca. 15 µm, which was 242 a good compromise for the overall size of the microstructure and the response needed for the 243 particular task of sper m release. In the same figure, the thermo -response of the final 244 microcarrier design in the swollen (at 25ºC), and the shrunken (at 40ºC) state of its microgate 245 is depicted (Video S3) . 246 Since the fabricated microcarriers are intended to be used in physiologi cal fluids, including the 247 oviduct fluid with viscoelastic properties, we tested the thermo -response of the chosen 248 microgate design in different media such as water, sperm medium (SP -TALP), and SP -TALP 249 enriched with 0.2% Methylcellulose (MC) (the latter mim icking the rheological properties of 250 ex vivo oviduct fluid in the luteal phase) (detail in Supplementary information; Figure 251 2d).[30] As expected, the thermo -response of the microgate was affected by the viscosity of the 252 10 medium, probably due to the different diffusion rate s within the polymeric -network of 253 PNIPAM.[49] According to Figure 2d , iii, the resulting shape change of the microgate at the 254 physiological ly relevant temperature range (from 35°C to 40°C) (detail of the chosen range 255 in the suppleme ntary information) and in the mimicking oviduct fluid (SP -TALP enriched 256 with 0.2% MC) was ca. 45 -60 µm2, which was sufficient for the sequential release of individual 257 sperm cell s. 258 259 Figure 2. a) Bulk temperature actuation test, (i) PNIPAM cubes with length of 30 µm and height of 10 260 µm in the swollen state (25°C) and shrunken state (40°C), (ii) Surface area changes of the cubes with 261 different writing parameters, red color shows higher cross -linked polymers /less temperature -response 262 11 (hard) and blue color show s lower cross -linked polymers /high temperature -response (soft) (n=1 per each 263 LP and SS, N=1 cycle) , (iii) Shape changes of the soft cube over time and temperature changes (25°C – 264 40°C) (n=1, N= 2 cycle s), b) Surface area changes of cubes with the length of 30 μm and height of 10 265 μm, 20 μm, and 30 μm, the yellow color is represented the high thermo -responsive PNIPAM cube (soft) 266 and the red color is represented the less thermo -responsive PNIPAM cube (hard), by increasing the 267 volume of PNIPAM, the shape changes a re more pronounced (n=1 per each LP and SS, N=1 cycle) , c) 268 (i–iii) Three different investigated designs for the microgate of the microcarrier in two different states, 269 swollen (25°C) and shrunken (40°C), and (iv) The fluorescence microscopy image of two exi sting 270 components of the microgate (red color represents PNIPAM and green color represents IPS) . Here 271 surface area change is the area indicated in transparent -red in Figure 2c.iii (n=1, N=2 cycle). d) 272 Temperature actuation test of the optimized gate design (c, iii) in different media : (i) water, (ii) SP - 273 TALP, and (iii) SP -TALP enriched with 0.2% MC . (n=1, N=2 cycle). The transparent -red region 274 highlights the physiological temperature range and the corresponding surface area change, which is then 275 used for the consequent experiments. 276 2.3. Microcarrier loaded with heparin for in situ sperm capacitation 277 For a successful fertilization process, sperm cells have to overcome different biological barriers 278 to finally reach the oocyte. Sperm cells have to be capacitated/hyp eractivated to be able to 279 penetrate the oocyte zona pellucida. Hyperactivation may also facilitate sperm cell release from 280 the oviductal storage reservoir.[50] In this regard, heparin has been widely used for sperm 281 capacitation in vitro , when the natural e nvironment is not available , as it induces biochemical 282 modifications that allow sperm cells to acrosome react upon exposure to the zona pellucida, 283 cumulus complex, and other substances associated to in vitro matured oocytes.[51] 284 As we aimed to facilitate s perm migration, and since PNIPAM can adsorb aqueous solution, 285 we loaded heparin into the PNIPAM microgates when they were immersed in TALP solution 286 (SP-TALP without bovine serum albumin (BSA) which triggers capacitation).[52,53] The loading 287 was carried out when the PNIPAM microgates were in the hydrophilic phase (swollen state , at 288 room temperature ) (detai ls in the Supplementary information) . The loaded heparin was then 289 released upon temperature increase (when PNIPAM shrank, the adsorbed aqueous -solution was 290 expelled from its polymeric network). Thus, the sperm cells which were in contact with the 291 heparin in the surrounded medium were locally capacitated/hyperactivated (conceptual image 292 shown in Figure 3a ). The fluorescence microscopy images (the bright f ield and fluorescence 293 channels) show the heparin loading into the PNIPAM microgate (Figure 3b) . 294 12 To estimate the concentration of loaded -heparin per each microcarrier and its effect on sperm 295 hyperactivation, we performed different experiments. First, the ef fect of different 296 concentrations of heparin (0, 0.025, 0.1, 0.25 mg mL-1 in SP -TALP) on sperm motion behavior 297 was tested. In agreement with Magdanz’s work, at 0.1 mg mL-1 of heparin, we obtained the 298 maximum average path and curvilinear velocity of sperm ce lls (Figure 3c , i).[54] The increases 299 in average path velocity in the presence of heparin were not significant because sperm 300 hyperactivation is followed by asymmetric tail beating with high amplitude and aggressive head 301 motion, which we observed by a prono unced increase in the curvilinear velocity. This 302 concentration was therefore considered optimal for subsequent experiments. 303 Next, we evaluated the effect of temperature on sperm mean velocity, as here the trigger to 304 release heparin from the PNIPAM microgat e was enacted by a temperature change, in contrast 305 to the aforementioned work, in which the trigger was enacted by a pH change (Figure 3c , ii). 306 By increasing the temperature from 35°C to 40°C (the physiological ly relevant temperature 307 range ), sperm velocity increased from ca. 34 to ca. 78 µm s-1 in sperm media without heparin 308 and from ca. 43 to ca. 103 µm s-1 in sperm media containing 0.1 mg mL-1 heparin, meaning a 309 17% increase in velocity in the presence of heparin. Here the most dominant effect on sperm 310 velocity came from temperature. Due to the physiological properties of sperm cells, described 311 by Rikmenspoel in 1984, sperm metabolism is accelerated by an increase in temperature, 312 altering both the frequency and amplitude of sperm tail beating.[55] The influence of temperature 313 on sperm velocity is reported in the previous work of our group.[28] In Figure 3c ,iii, one can 314 see the capacitation/hyperactivation of both parameters, temperature and heparin, in the 315 increased number of sperm cells which swa m in circular trajectories. Moreover, sperm motility 316 was also investigated (Figure S2). As expected, at a higher temperature, sperm cells lose their 317 motility faster than at a lower temperature because of the higher swimming speed and energy 318 consumption rat e (Figure S 2a). This reduction was even more pronounced when the sperm 319 cells were capacitated/ hyperactivated (Figure S 2b). Thus, it can be assumed that 320 hyperactivation has a significant effect on sperm motility by increasing the rate of energy 321 consumption and therefore reducing their lifetime. 322 After loading the microcarriers with heparin, a quantitative toluidine blue assay was carried out 323 to determine the concentration of heparin per microcarrier (detail in Supplementary 324 information) . As shown in Figure 3d , ii, by increasing the temperature, PNIPAM was 325 gradually shrunk, enabling heparin release. While maintaining a constant temperature of 40°C, 326 heparin release was measured at different time intervals. After 15 min there was no change in 327 13 the absorbance value , indicating that no further heparin was released (Figure 3d , iii). According 328 to the absorbance results and the calibration curve in Figure 3d , i, the concentration of loaded 329 heparin per microcarrier was calculated to be 0.465 pg. 330 Finally, the effect of t he microcarrier -released heparin on the surrounding sperm cells was 331 investigated (Figure 3e) . In the first set of experiments (left columns) in Figure 3e.i , on sperm 332 solutions with and without 0.1 mg mL-1 heparin, the increase in sperm velocity followed th e 333 same trend as shown in Figure 3c . At physiological temperature , the sperm velocity increased 334 2.3 times when the sperm solution contained 0.1 mg mL-1 of heparin. In the second set of 335 experiments (right columns), we measured the sperm velocity in the prese nce of microcarriers 336 with and without heparin (free sperm cells in the solution were considered). When the 337 microcarriers were not loaded with heparin, the sperm velocity was similar to that of the free - 338 swimming sperm cells without heparin. In the case of m icrocarriers loaded with heparin, as the 339 temperature was raised to trigger the release of heparin, sperm velocity increased (Figure 3e , 340 i). Consequently, in Figure 3e , ii, the effect of heparin on the motion trajectory of the sperm 341 cell was demonstrated. The motion trajectories of sperm cells at room temperature (25°C) and 342 physiological temperature (3 8°C), with and without heparin in the solution, reveal the fact that 343 both temperature and heparin have a positive effect on the sperm capacitation/hyperactiva tion. 344 The hyperactivated sperm cells are intended to swim in a circular motion pattern because of an 345 increase of amplitude and asymmetrical tail beating (Video S4 ). 346 14 347 Figure 3. a) Conceptual schematic of heparin release and sperm hyperactivation, b) Fluore scence 348 microscopy images show the presence of heparin loaded in the PNIPAM microgates, red channel 349 represents the PNIPAM gates (presence of RhB) and green channel represents the loaded heparin, c) (i) 350 Curvilinear and average path velocity of free sperm cel ls in different concentrations of heparin in SP - 351 TALP (n=200, N=3, mean, ±SD), (ii) Effect of 0.1 mg mL-1 heparin on sperm velocity at different 352 temperatures (n=200, N=3, mean, ±SD), (iii) Percentage of sperm cells with circular trajectories with 353 and without the presence of heparin in SP -TALP (n=200, N=3, mean, ±SD), [analyzed by SCA® CASA 354 System for semen analysis], d) (i) Calibration curve of heparin based on toluidine blue assays, and 355 Accumulative heparin release from microcarriers at (ii) different temperatures (nmicrocarrier =1500, N=3, 356 15 mean, ±SD), and (iii) time durations (nmicrocarrier =1500, N=3, mean, ±SD), [absorb ance at 631 nm ], e) 357 (i) Effect of released heparin from microcarriers on sperm velocity at room temperature (25°C) and 358 physiological t emperature (38°C) (n=200, n microcarrier =20, N=3, mean, ±SD), [analyzed by SCA® CASA 359 System for semen analysis] , (ii) The motion trajectory of non -hyperactivated and hyperactivated sperm 360 at room temperature and physiological temperature with and without heparin in the solution. In all 361 experiments containing sperm, we used washed sperm which had not undergone any pr eselection 362 process, e.g., swim -up, to avoid any sperm capacitation and only monitor the effect of heparin. 363 2.4. Microcarriers decorated with hyaluronidase post -loaded polymersomes (HYAL - 364 Psomes) for in situ degradation of the HA -cumulus complex 365 Hyaluronidase (H YAL) is a hyaluronic acid -containing sperm acrosomal enzyme that plays an 366 important role in the fertilization process through its participation in the degradation of the 367 extracellular matrix (ECM) of the cumulus cells.[56] In in vitro fertilization (IVF) a nd 368 intracytoplasmic sperm cell injection (ICSI), HYAL is used to remove the cumulus cells around 369 the oocytes.[35] We intended to address infertility -related lack of HYAL, caused by low sperm 370 count, by adding HYAL onto the microcarrier in order to help spe rm cells digest the cumulus 371 cells—the final barrier to fertilization. For this purpose, we proposed a novel nanocarrier 372 platform based on polymersomes (Psomes) for post -loading of HYAL, as Psomes are further 373 immobilized onto the microcarrier’s surface. Pso mes are prepared based on the self -assembly 374 of amphiphilic block copolymers which provide them with excellent mechanical and chemical 375 stability. Moreover, Psomes’ membrane permeability can be triggered by external stimuli (e. 376 g., pH, temperature, redox po tential, light, magnetic field, and ultrasound) .[57] This platform 377 allowed us to uniformly immobilize enzymes onto the microcarrier’s surface while preserving 378 their activity. 379 2.4.1. HYAL -Psomes preparation and characterization 380 First, pH -responsive and photo -crosslinked Psomes were synthetized by self -assembly of block 381 copolymers (BCPs) using the so -called pH switch method (Figure S 3; detail in 382 Supplementary information) .[58–60] The chemical structure, composition, molar mass and 383 dispersity of the BCPs used are listed in Table S1 . Then, hyaluronidase was integrated into the 384 membrane of the Psomes using a recently established protein post -loading approach (Figure 385 4a; detail in Supplementary information) .[61] Here, different charact erization approaches 386 were conducted to measure loading efficacy, such as the particle shape, size, and membrane 387 thickness of Psomes before and after enzyme loading (Empty -Psomes, and HYAL -Psomes) 388 16 (Table 1) . We employed cryogenic transmission electron micro scopy (cryo -TEM) and 389 compared the results with dynamic light scattering (DLS) measurements. As one can see in 390 Table 1 , the increase in diameter confirms the successful loading of HYAL; however, there is 391 no difference in membrane thickness, for which two ex planations are proposed: either the 392 enzyme is attached to the Psomes’ surface, or it is too small to change the membrane 393 thickness.[60] Furthermore, the pH cycles and pH titration of Empty -Psomes and HYAL -Psomes 394 with DLS were also considered to check rever sible pH -response and the swollen/collapsed 395 states of Psomes before and after the loading process (Figure S 4; Table S2) . 396 Table 1. Diameter and membrane thickness of Empty -Psomes (before post -loading) and HYAL -Psomes 397 (after post -loading) with cryo -TEM and DLS. ( 𝐶𝑃𝑠𝑜𝑚𝑒𝑠 = 0.5 mg mL-1, 𝐶𝐻𝑌𝐴𝐿 = 0.2 mg mL-1, 1 mM 398 PBS) at pH 8. DLS measurements were carried out in triplicate. Calculated values using cryo -TEM 399 images were obtained by analyzing 60 particles. 400 Empt y-Psomes Before post -loading HYAL -Psomes After post -loading Diameter by DLS [nm] 78.0 ± 0.9 85.1 ± 1.8 Diameter by cryo -TEM [nm] 70.8 ± 7.2 82.6 ± 4.9 Membrane thickness [nm] 14.0 ± 1.1 14.0 ± 0.8 To estimate the loading efficiency of HYAL -Psomes , we used labeled enzymes (here we used 401 RhB) (Figure S 5) with the same post -loading process and preparation of the solution as naked 402 enzymes (detail in Supplementary information) . The calibration curve of the used RhB -SCN 403 is depicted in Figure S 6. The load ing efficiency was carried out after the purification process 404 of RhB -HYAL -Psomes (during the dialysis process, unloaded RhB -HYAL was defused out 405 because of its smaller size) (Figure S 7a, i). Next, the absorbance of the solutions per 406 purification day w as measured (the absorbance peak correlated to the amount of RhB in the 407 solution). The maximum loading efficiency was ca. 41% (Figure S 7a, ii), and the average 408 loading efficiency for at least 10 experiments was ca. 28% (Figure S 7a, iii). 409 The enzymatic activi ty of HYAL -Psomes was also evaluated after purification, and considering 410 different storage conditions via optical density measurement (Figure S 7b; detail in 411 Supplementary information) . For this purpose, the enzymatic activity was compared with the 412 calibrat ion curve of fresh free HYAL and unpurified HYAL -Psomes (Figure S 8). The residual 413 enzymatic activity of HYAL -Psomes after 72 H was approximately 20%, which agrees with 414 the above -mentioned loading efficiency experiments estimated by fluorescence spectroscop y. 415 In the same graph, the enzymatic activity of HYAL -Psomes under different storage conditions 416 17 (fridge (4°C), and freezer ( -20°C)) is also depicted (Figure S 7b). Based on these results, both 417 storage methods, fridge and freezer, can be recommended as promis ing long -term storage 418 condition. 419 Finally, HYAL -Psomes showed satisfactory stability under pH changes which naturally occur 420 in the female reproductive tract (pH ~ 4.4 in the vagina, pH ~ 7-7.2 in the uterus, pH ~ 7.9 in 421 the oviduct) (Figure S 7c, i), with n o significant HYAL release (Figure S 7c, ii). Thanks to this 422 characteristic, HYAL -Psomes are suitable candidates to mediate degradation of the HA - 423 cumulus complex, with the vision of facilitating sperm journey to the oocyte in future in vitro 424 and in vivo fer tilization experiments.[62,63] 425 2.4.2. Immobilization of HYAL -Psomes on the microcarrier’s surface 426 The immobilization of HYAL -Psomes on the microcarrier surface based on a non -covalent 427 interaction between β -cyclodextrin (β -CD) (on the microcarrier surface) and polyethylene 428 glycol (PEG) chains of Psomes, was carried out as shown in Figure 4b (detail in 429 Supplementary information) . To con firm Psomes immobilization onto the microcarrier 430 surface, different characterization methods were employed. In the confocal laser scanning 431 microscopy (CLSM) images, in which we used RhB -HYAL -Psomes for better visualization, 432 one can see that the microcarrie r surface was uniformly decorated by RhB -HYAL -Psomes 433 (Figure 4c) . To validate those results, SEM imaging was performed, in which we observed a 434 rough and heterogeneous surface of the functionalized microcarrier due to the presence of the 435 HYAL -Psomes, compar ed to the smooth surface of the non -functionalized microcarrier (Figure 436 4d). The cryo -TEM images in Figure 4e show spherical objects representing Empty -Psomes 437 and HYAL -Psomes on the microcarrier surface with a quite uniform shape and size following 438 immobil ization. 439 Finally, as a proof of concept, the interaction between HYAL -Psomes and isolated cumulus 440 cells (HA -cumulus complex) was investigated (Figure 4f) . The first two images show only the 441 HA-cumulus complex in the IVF medium after 30 min of incubation at 38°C (in in vitro 442 fertilization of bovine gametes, it is recommended to expose cells to high concentrations of 443 hyaluronidase (50 -250 IU mL-1 ~ 0.06-0.3 µg mL-1 for ca. 30 mi n).[64] There was no obvious 444 evidence of HA -cumulus complex degradation after 30 min of incubation. The second 445 experiment was conducted with 0.04 µg mL-1 hyaluronidase in the IVF solution, in which the 446 cumulus cell cluster disintegrated into small pieces after 30 min of incubation. As a third 447 experiment , we considered non-loaded microc arriers with HYAL -Psomes (ca. 50 448 18 microcarriers). In that experiment, there was no evidence of HA -cumulus complex 449 disintegration as in the previous experiments. Finally, in the last experiment, ca. 50 450 microcarriers immobilized with HYAL -Psomes were put toge ther with the cumulus cells, 451 resulting in disintegration of the cell cluster into small pieces after 30 min of incubation (Figure 452 S9) (more experiments with murine and bovine cumulus complex w ere added in 453 Supplementary information ). 454 In this qualitative stu dy, we can observe an interaction between immobilized HYAL -Psomes 455 on the microcarrier surface and HA -cumulus complex which can be used as local HA digestion 456 in future in vivo scenarios, but further studies still need to be carried out with oocytes and unde r 457 more realistic conditions, also considering the mechanical removal of cumulus cells by the 458 action of post -capacitation sperm tail beating. 459 19 460 Figure 4. a) Schematic of the fabrication process of HYAL -Psomes, b) Schematic of the immobilization 461 of HYAL -Psomes on the microcarrier surface for local degradation of HA -cumulus complex, c) CLSM 462 characterization of immobilized microcarrier with RhB -HYAL -Psomes (in brightfield, fluorescence, 463 and merged channel), d) SEM characterization of immobilized microcarrie r with and without HYAL - 464 Psomes, [the inset images show the respective surface in more detail], e) Cryo -TEM images of 465 immobilized Empty -Psomes and HYAL -Psomes on the microcarrier surface (exemplary Psomes 466 marked with yellow arrows) , f) Qualitative study of the interaction of hyaluronidase with HA -cumulus 467 20 complex (with and without hyaluronidase in the solution and with and without HYAL -Psome loaded 468 microcarriers). 469 2.4.3. Effect of HYAL and HYAL -Psomes on sperm motility 470 In Figure S2c , it is observed that sperm motility decreases in all conditions after 6h of 471 incubation at 38°C. The decline of sperm motility in the case of pure sperm solution and sperm 472 solution in presence of hyaluronidase (HYAL) was ca. 86%, and 75%, respectively. Likewise, 473 sperm solution contai ning Empty -Psomes and HYAL -Psomes, showed a motility decrease of 474 90%, and 70%, respectively. In both cases, we could observe that sperm sample containing 475 either HYAL solution or HYAL -Psomes, exhibited a higher motility rate than the samples 476 without HYAL. T his might be due to the fact that HYAL and other enzymes present in the 477 sperm acrosome play an important role in the preparation of sperm prior oocyte fertilization. 478 Hyaluronidases in particular are responsible of catalyzing the hyaluronic acid present in the 479 cumulus complex.[65] In general, there is a complex cascade of reactions associated with 480 changes in the composition of the sperm membrane during the sperm journey, some of them 481 triggered by hyaluronidases, which has a direct influence on sperm motility. However further 482 studie s need to be realized to better understand the underlying mechanism and correlation 483 between sperm motility, and a particular enzyme activity. 484 In addition, for a better understanding of the mutual effect of HYAL -Psomes and heparin on 485 sperm motility, we perf ormed combinatory experiments, involving both HYAL -Psomes and 486 heparin, mimicking the envisioned in vivo scenario. In Figure S2d , sperm motility was 487 measured in solution in presence of heparin with HYAL, heparin with Empty -Psomes, and 488 heparin with HYAL -Psom es. As discussed in Figure S2c , the presence of HYAL in the solution 489 had a positive effect on sperm motility. In Figure S2d , the same effect was observed for 490 hyperactivated sperm cells when there was HYAL in the solution, the sperm motility was 491 preserved s ignificantly compare d to the pure sperm solution (improvement of sperm motility 492 ca. 40% for hyperactivated sperm cells in presence of HYAL, and ca. 66% for hyperactivated 493 sperm cells in presence of HYAL -Psomes). 494 In the case of hyperactivated sperm cells in presence of polymersomes (Empty -Psomes and 495 HYAL -Psomes), we observed better preservation of sperm motility. According to the 496 previously reported work from Deng and coauthors, polymersomes can react with reactive 497 oxygen species (ROS).[66,67] ROS are caused by oxidative stress that sperm experience during 498 manipulation, reducing their motility. Therefore, and based on the obtained results, 499 21 polymersomes possibly react with oxygen species in the solution, removing them and 500 preserving the motility of hyperactiva ted sperm cells for a longer time. As heparin and 501 hyaluronidase activity affect the composition of the sperm head membrane differently, it is 502 expected that sperm motility is also influenced, however the underlying physicochemical 503 effects by the interplay o f both heparin and hyaluronidase, as well as, of other enzymes present 504 in the sperm acrosome, require deeper investigations. 505 2.5. Sperm -microcarrier motion performance in oviduct -mimicking fluids 506 As explained above, the microcarrier has ca. 16 available caviti es for the coupling of sperm 507 cells; however, due to possible tail beating interference, we observed coupling of up to 10 sperm 508 cells in our experiments (an example of multiple sperm cells coupled into a microcarrier is 509 shown in Figure 5a , with sperm coupli ng pointed out by the yellow arrow) (Videos S5) . In 510 Figure 5b , the trajectories of the optimized sperm -microcarrier are shown in different media 511 (SP-TALP, and SP -TALP enriched with 0.2% MC). The latter shows the same rheological 512 behavior as the early lutea l phase of bovine oviduct fluid BOF -EL (the phase after ovulation 513 where fertilization takes place).[30] The mean velocity of 20 sperm -microcarriers showed 50% 514 decrease in the measured velocity from SP -TALP to SP -TALP enriched with 0.2% MC, which 515 agrees wit h previous results (Figure 5c; Video S6 and Video S7 ).[30] 516 2.5.1. Capture, transport, and release of sperm cells 517 Another challenge which should be considered regarding the biomedical application of such 518 microcarriers is their controllability. When motile sperm cells propel the synthetic microcarrier 519 and push it forward, by applying external stimuli such as a non -invasive and biocompatible 520 magnetic field, the carrier can be guided toward a desirable location. In this work, the 521 incorporation of a thin film of iron was included for the magnetic actuation. An external 522 magnetic field was used for the guidance of microcarri ers which were propelled by motile 523 sperm cells. After completion of sperm cell coupling into microcarriers, they were steered to 524 the desired location. In all these experiments, a permanent neodymium magnet with a magnetic 525 field flux of ca. 20 mT at the loc ation of the sample was used (Figure S 10). 526 To demonstrate the whole process of capture, transport, and release of multiple motile sperm 527 cells with microcarriers, a microfluidic system containing three reservoirs made of 528 polydimethylsiloxane (PDMS) was used (Figure 5d; detail in Supplementary information) . 529 Before the experiment, the microchannel and microcarriers were treated with Pluronic acid for 530 22 30 min, and washed with distilled water. Then the microcarriers were released from their 531 substrate with a suffi cient quantity of SP -TALP. Subsequently, sperm solution (swim -up 532 process -selected sperm cells immersed in fresh SP -TALP) was added to the microchannel 533 (detail in Supplementary information) . After sperm coupling, sperm -propelled microcarriers 534 in the first r eservoir were guided through the microchannel by means of an external magnetic 535 field, reaching a second reservoir. Next, their orientation was altered by the external magnetic 536 field to ultimately reach the third reservoir, where sperm release was demonstra ted (Figure 5d) . 537 During the whole transport, the temperature was fixed at 35°C using a Peltier element under 538 the petri dish. After reaching the last reservoir, the temperature was increased to 40°C. The 539 PNIPAM microgate opened to an appropriate size to ena ble sperm to be released one at a time 540 (Figure 5e) . The whole process of capture, transport, guidance, and release of at least 5 sperm 541 cells is shown in Video S 8. Another example of the whole process can be seen in Video S 9. 542 Furthermore, we analyzed the performance of the developed sperm -microcarriers on ex vivo 543 fallopian tube tissue. For this experiment, we fabricated a double -layer parafilm channel (height 544 ~ 1mm), in which we placed a flat piece of ex vivo oviduct tissue (laying down on the bottom 545 surface), and filled the whole chamber with sperm medium (SP -TALP). Then we introduced a 546 small drop containing sperm cells and microstructures. Once the structures were coupled and 547 pushed forward by the sperm cells, we used an external magnetic field (ca. 20 mT at the location 548 of the sample) to guide the sperm -microcarriers toward the tissue. We did two types of imaging, 549 one employing an upright microscope (reflective microscope), where we could observe the 550 moving sperm -microcarriers swimming on top and un derneath the tissue. We confirm the 551 presence of the sperm microcarriers by fluorescence microscopy . The second imaging was done 552 with an inverted microscope, to see sperm -microcarriers swimming below the tissue, between 553 the bottom glass substrate and the ov iduct tissue, or by flipping the parafilm channel to see the 554 ones on top of the tissue when the light from behind illuminates it. As shown with fluorescence 555 microscopy, after a certain time, the microcarrier swimming on top of the tissue started to get 556 stuck (Video S 10 and Video S11 ). For that further investigations on surface functionalization 557 and protein -repellent coatings need to be realized to ensure optimal performance when 558 employing them in living organisms. For instance, Wu and coauthors reported the helical 559 micropropeller that can penetrate the vitreous humor in the eye which was functionalized with 560 a perfluorocarbon surface coating to minimize the interaction of the microrobots with tissues 561 including collagen bundles.[68] In another work, Cabanach a nd coauthors proposed non - 562 23 immunogenic stealth zwitterionic microrobots that avoid detection and phagocytosis from 563 macrophages.[69] 564 2.6. Microcarriers as enhanced contrast agents for photoacoustic imaging 565 Finally, as the microcarriers were also coated with a th in film of gold (20 nm of Au), they could 566 absorb infrared (IR) light and convert it into acoustic waves, which is beneficial for their 567 visualization using a dual ultrasound (US) and photoacoustic (PA) imaging system under 568 scattering tissues for future in vivo applications. PA imaging provides the penetration depth of 569 US and the molecular specificity of optics, and has successfully been implemented in in vivo 570 imaging of micromotors.[70] The setup is equipped with a linear array of US transducer and fiber 571 optic bundles on either side of the transducer for illumination (680 to 970 nm), as shown in 572 Figure 5f . 573 To observe the dynamic behavior of the microcarriers in the imaging system, a tra nsparent 574 intravascular polyurethane (IPU) phantom tube was used (Figure 5g; detail in Supplementary 575 information) . The dynamic imaging of a cluster of microcarriers is shown in time -lapse images 576 from 0 to 2.80 s (Figure 5h , i), comparing optical, US, and PA imaging modes. The dashed - 577 circle shows the position of the cluster of microcarriers at starting position A and final position 578 B after travel. The magnetic guidance was achieved using a hand -held permanent magnet placed 579 close to the phantom tube. It was po ssible to image a cluster of microcarriers with absorbing 580 metal layers coated on the surface. Such metal layers provided strong PA amplitude values as 581 compared to the control (with no microcarriers) in comparable imaging regions (Figure 5h , ii). 582 According to Figure S 11, it is observed that the employed illumination during photoacoustic 583 imaging is not sufficient to induce local heating (by photothermal effect) in the sperm 584 microcarriers, which can undesirably cause a shape change in the microcarrier PNIPAM 585 microgate (detail ed characterization and calculations are included in the Supplementary 586 information) . 587 24 588 Figure 5. a) Sperm coupling into a microcarrier cavity, the yellow arrow shows the position of the sperm 589 head, b) Sperm -microcarrier trajectory in the SP -TALP, and SP -TALP enriched with 0.2% of MC, c) 590 The sperm mean velocity in SP -TALP and SP -TALP + 0.2% MC (nmicrocarrier =20, N=3, mean, ±SD), d) 591 Schematic of microfluidic channel to demonstrate sperm capture, guidance/transport, and release with 592 the sequential figures (the red arrow shows sperm -microcarrier location), e) The sequential images of 593 sperm release at physiologically relevant temper ature ca. 39°C (the red arrow shows the position of the 594 sperm head), f ) Schematic showing the working principle of the employed hybrid US and PA imaging 595 system, g) Schematic of a swarm of microcarriers in a phantom tube immersed in a water bath for real - 596 25 time tracking of their movement, h) (i) Time -lapse images of moving microcarriers in the bright field, 597 US , and PA modes (yellow dashed -circle shows the cluster of moving microcarriers at starting position 598 A and final position B), (ii) PA amplitude values ex tracted from the captured PA images of moving 599 microcarriers, compared to the control sample (the tube with no microcarriers) (n=2, N=3, mean, ±SD). 600 3. Conclusion and Outlook 601 In the current work we focused on assisting sperm cells when sperm count is very low (also 602 called oligospermia ) in order to increase the chances of oocyte fertilization in vivo. For this 603 reason, we fabricated multifunctional sperm -hybrid microcarriers able to assist a limited 604 number of sperm cells. First by realizing the in -situ sperm capacitation/hyperactivation by the 605 local release of heparin. Second by triggering the sperm release on the desired location, and 606 finally by facilitating the removal of the cumulus complex that surrounds the oocyte to open a 607 path for the sperm cells to reach the oocyte zona pellucida. 608 For that purpose, a stream -lined microstructure with a stimuli -responsive “microgate” was 609 fabricated. The microgate was loaded with heparin prior sperm capture. The optimum 610 concentration of heparin per microcarrier was calculated and the effect of the heparin on sperm 611 hyperactivation was also investigated. We confirmed that temperature in combination with 612 heparin has a significant effect on sperm hyperactivation as both increase sperm metabolism 613 and affect their motion performance. 614 Additionally, the microcarriers were functionalized with surface -active hyaluronidase -loaded 615 polymersomes, which enable a high loading efficiency while preserving their enzymatic activi ty. 616 We demonstrate that the enzyme was stably loaded onto the polymersomes under varying pH, 617 allowing for local enzymatic reactions and facilitating the digestion of the extracellular matrix 618 of the cumulus cells via hyaluronic acid surrounding the oocyte. 619 We also show that such microcarriers containing ca. 10 sperm cells can be steered in a targeted 620 fashion by means of an external magnetic field, which was demonstrated in a confined 621 microfluidic channel using an artificial oviduct fluid. We successfully rep ort the capture, 622 transport, and release of multiple sperm cells by the designed microcarriers. The 623 microstructures were coated with Au, enabling preliminary bioimaging tests to be conducted 624 using photoacoustic imaging. The microstructures’ photoacoustic signal was obtained by 625 employing phantom plastic tubes while moving, demonstrating a possibility to track them under 626 scattering tissues for future in vivo applications. 627 26 Despite these important achievements, compared to previously reported single -sperm carrie rs, 628 there are still many challenges to be addressed before the microcarriers can be applied in vivo . 629 For example, these microcarriers were designed and proved using bovine sperm. However, in 630 order to perform animal trials, and due to the current limitation s of imaging techniques in terms 631 of spatiotemporal resolution and penetration depth, small animal models (e.g., mice) are 632 preferable for establishing the technology , and for that reason will be considered in the future . 633 The coupling and transport of multi ple microcarriers, where each contains up to 10 sperm cells, 634 might be sufficient to demonstrate oocyte fertilization, but their cooperative action require 635 further optimization. Also, the interactions of such a microcarrier with the surrounding tissue 636 remai ns to be studied. We have so far evaluated their performance in artificial oviduct fluids 637 with similar rheological properties, but the interaction with the oviduct epithelial and ciliary 638 cells is the aim of future studies by our group; before initiating an imal trials, the 3Rs principle 639 (reduction, refining, and recycling) should be followed and supported by a complete set of 640 experiments performed in vitro and with ex vivo tissue .[71] Furthermore, these structures should 641 ideally be biodegradable. There are biodegradable photoresists and reported protocols for 642 employing photo -patternable gelatin currently available, which might be used in place of IPS 643 photoresist.[72,73] Gelatin is also responsive to pH and temperature changes, and current efforts 644 are being made to develop biodegradable stimuli -responsive hydrogels.[54,74,75] 645 All in all, we have addressed several challenges reported in the literature: namely, the transport 646 of multiple sp erm cells, their imaging for future in vivo applications, and the addition of 647 multiple functions (sperm capacitation and cumulus cell removal). To accomplish these aims, 648 we employed different functional materials to fabricate microcarriers with adaptable s timuli - 649 responsive microgates for different operations. Such stimuli are already present in the fallopian 650 tube, which will circumvent the need for external triggering in future in vivo experiments. This 651 advanced 4D -printed multifunctional microcarrier made with two -photon lithography, offers a 652 plethora of possibilities not only in the field of microrobotics -assisted therapy and diagnosis 653 but also for on -chip applications. 654 Acknowledgements 655 Authors thank Ms. Franziska Hebenstreit for conducting the cell cultur e experiment and 656 bringing bovine ovary from slaughterhouse along with Mr. Friedrich Striggow to collect 657 cumulus cells , Ronald Naumann , from MPI -CBG for providing the murine gametes samples, 658 Mr. Lukas Schwarz and Mr. Haifeng Xu for the initial support on th e microcarrier design. Mr. 659 27 Friedrich Striggow and Dr. Sandhya Rani Goudu for conducting internal revision of the paper, 660 Ms. Franzisca Kucharczyk for performing the fluorescence spectroscopy, Mr. Yang Zhou for 661 performing Cryo -TEM, and Mr. Michael Göbel for preparing SEM images. 662 Funding 663 This work is part of the projects that have received funding from the European Research Council 664 (ERC) under the European Union’s Horizon 2020 research and innovation program (grant 665 agreement nos. 835268 and 853609). This work was supported by the German Research 666 Foundation DFG (Gottfried Wilhelm Leibniz Prize granted in 2018, SCHM 1298/22 -1 and 667 KA5051/1 -1). 668