Polymeric vesicles represent a promising candidate for usage in drug delivery systems due to their facile assembly and ability to provide a stable soft interface. Among these materials, polyethylene glycol-polystyrene block copolymers (PEG-PS) stand out for their versatility and adaptability. These copolymers exhibit a remarkable propensity for self-assembly, allowing the formation of vesicles capable of undergoing diverse shape transformations. Notably, they can adopt the distinctive stomatocyte morphology, characterized by a concave shape with a central cavity . Such structures hold significant potential for drug delivery applications as nanomotors, offering both encapsulation capability and controlled release functionalities . Moreover, researchers have explored novel shapes utilizing PEG-PS copolymers, including the growth of protrusions from the vesicle surface . While these emerging shapes warrant further investigation to elucidate their optimal utilities, their creation underscores the material's versatility in shape manipulation. This ability to engineer a spectrum of shapes from a single material holds profound implications for drug delivery and beyond. By tailoring vesicle morphology to specific requirements, researchers can optimize drug encapsulation, targeting, and release, advancing the efficacy and precision of therapeutic interventions. Continued exploration of these versatile materials promises to unlock new avenues in pharmaceuticals and biomedical engineering. The problem here is that PEG-PS is a non-biodegradable material and this poses significant challenges for its use in drug delivery applications, particularly concerning potential accumulation in the body . Recent advancements have sought to address this issue by replacing the hydrophobic block with biodegradable alternatives, like polylactic acid, resulting in PEG-PDLLA block copolymers capable of forming vesicles that are able to undergo shape transformation towards stomatocytes . Despite this progress, the persistence of the non-degradable PEG segment is still ongoing as PEG is regarded as the benchmark for hydrophilic polymers used in drug delivery .

The non-biodegradability of PEG under most conditions, coupled with recently discovered immunogenic responses to it, has led to increasing concerns . This debate has prompted exploration into novel materials that resemble PEG, retaining its positive attributes, but add biodegradability. Two of such classes of polymers that would form viable alternatives are polyoxazolines and polypeptides , both of which possess biodegradable and biocompatible properties. Moreover, these materials offer versatility in their synthesis, allowing for the incorporation of various building blocks to tailor the polymers to desired specifications. Additionally, they lend themselves well to the synthesis of block copolymers, further expanding their potential applications for the use in drug delivery systems .

In this regard, synthetic polypeptides emerge as a promising candidate for constructing biodegradable and biocompatible polymersomes. Leveraging the inherent presence of peptide-degrading mechanisms within the human body and the versatile chemical functionalities of naturally occurring amino acids, synthetic polypeptides offer a robust platform for designing drug delivery systems that meet the criteria of biodegradability and biocompatibility . The present study focuses on a polysarcosine and poly-ʟ-(benzyl glutamate) block copolymer (PSar-PBLG), as it is known to be able to self-assemble in a variety of structures, including micelles, vesicles, and nanoparticles . However, the versatility of these polymers in shaping vesicles into asymmetric structures suitable for nanomotors remains relatively unexplored. While previous research has demonstrated their ability to form round vesicles and some tubular shapes, the pursuit of asymmetric structures, such as the stomatocyte, presents a novel challenge and opportunity in the field .

The stomatocyte shape would be an excellent addition because of its demonstrated suitability for nanomotor fabrication . The necessary shape transformation for achieving this morphology is primarily driven by changes in osmotic pressure. Initially achieved through dialysis and later by the addition of PEG solution , the process involves deflating the polymeric vesicle, prompting it to bend and adopt the stomatocyte configuration. Several critical factors contribute to this transformation. Firstly, the application of osmotic pressure which must be sufficiently robust. This control is notably easier with the addition of PEG, as it swiftly creates a substantial osmotic gradient . Secondly, the vesicle's properties are crucial, particularly its permeability. Excessive permeability allows water molecules – and potentially larger molecules – to traverse the membrane without exerting adequate force, undermining the transformation process . Lastly, membrane stiffness plays a role; the stiffness of the membrane determines how it responds to the applied force. Besides that stiffness should be sufficient to maintain membrane curvature for a shape transformation to occur . This is particularly important when domains are formed over the membrane with different stiffness which can lead to an asymmetrical response . An excessive stiffness will eventually even impede deformation entirely . This characteristic is harnessed in systems to stabilize shapes by water addition, effectively locking the membrane in a kinetically stable state .

In a recent study by Elafify, M. S. et al. it was demonstrated that polysarcosine-based self-assemblies could undergo shape transformation via dehydration induced by heating . This dehydration of the polysarcosine caused in their system a complete shape transformation but it would effectively also reduce membrane permeability and enhance osmotic pressure, as temperature is a factor in the van ‘t Hoff formula for osmotic pressure . Additionally, dehydration strengthens polysarcosine chains interactions , potentially rendering the membrane too rigid for shape transformation. This means that the heating of the vesicles made with polysarcosine would improve the effectivity of the osmotic pressure applied up to a certain point, after which the membrane would be too rigid for deformation. Within this window it is expected that stomatocytes could be obtained using PSar-PBLG block copolymers.

Scheme 1: i) Synthesis of benzyl glutamate NCA using phosgene and propylene oxide as a scavenger. ii) Ring-opening polymerization of BnGluNCA using benzylamine as initiator. iii) Synthesis of sarcosine NCA starting from Boc-protected sarcosine using phosgene and propylene oxide as a scavenger. iv) Ring-opening polymerization of Sar NCA using the benzyl glutamate homopolymer as macro initiator.

Figure 1: i) The PBLG-PSar block copolymers are dissolved in DMF and then assembled though the solvent-exchange method by addition of 66% water. ii) The membrane is dehydrated at 70 °C for 1 min before starting the shape transformation. iii) The shape transformation is induced by osmotic shock. This is done by the addition of 5 µL PEG2000 solution (400 mg/mL). The osmotic shock can also be induced by PSar solution or saccharose solution.

Figure 2: Progression of shape transformation of PSar-PBLG vesicles at 70 °C (scalebar 0.5 µm). a) Initial (0 s): polymersome formation. b) Transition (30 s): formation of a vesicle with a dent and stomatocytes. c) Further shape transition (60 s): to a wide-open stomatocyte. d) Final (120 s, angle 35°): shape evolved into a disk after 120 s before quenching.

Figure 3: The temperature-dependent behavior of vesicles and shape Transformation: a) Thermograph of PBLG-PSar: Showing dehydration with a peak around 70 °C. b) The fluorescent intensity of pyrene measured at 376.6 nm divided by the intensity at 387.6 nm tracked over temperatures between 20 °C and 80 °C. c) DLS measurement: demonstrating a slight decrease in hydrodynamic radius of the vesicles at higher temperatures. d–f) Vesicles after shape transformation: illustrated at 60 °C, 70 °C, and 80 °C, respectively.

Self-assembled vesicles crafted from polysarcosine-poly(benzyl glutamate) block copolymers exhibit distinct properties compared to conventional systems like PEG-PS or PEG-PDLLA, notably their inability to undergo shape transformation though osmotic pressure under conventional conditions. This limitation arises from the inherent membrane flexibility and heightened permeability of these vesicles. However, polysarcosine exhibits a dehydration temperature between 40 °C and 90 °C, triggering the release of water molecules from the hydrophilic layer. Consequently, enhanced interactions between polysarcosine chains ensue, culminating in a more rigid and less permeable membrane structure at an optimum dehydration at 70 °C.

This augmented membrane permeability facilitates the buildup of stronger osmotic pressure within the vesicle, driving deformation into a stomatocyte shape. This novel morphology for polysarcosine-based polymers opens new avenues for utilizing fully biodegradable polymers as nanomotors, leveraging enzyme encapsulation techniques. Moreover, it was observed that a temperature that is too high does not yield further shape transformation but instead results in the formation of more rigid polymersomes. So, the system only operates accordingly in a narrow window.

Lastly besides the PEG addition method for inducing osmotic pressure most hydrophilic molecules should be able to induce enough osmotic pressure to induce shape transformation. The use of saccharose which has a higher dehydration temperature then PEG or PSar seems to be more effective for use at high temperatures.

In summary, the distinctive properties of polysarcosine-poly(benzyl glutamate) block copolymers pave the way for innovative applications in drug delivery and nanotechnology, marking a significant advancement in the field of biodegradable polymer vesicles. Further exploration into optimizing shape transformation conditions tailored to one’s goal and harnessing the unique capabilities of these vesicles holds promise for future biomedical applications.