Introduction

Poly(β-l-malic acid) (β-PMLA) is a polymeric material formed by ester bonds between the hydroxyl and β-carboxyl groups of l-malate [1, 2]. Only β-PMLA is naturally available when produced by certain microorganisms, mainly Aureobasidium pullulans (A. pullulans) and Physarum polycephalum (P. polycephalum), whereas all α-, β-, and α, β-PMLAs can be chemically synthesized (Fig. 1). Although malic acid conforms in both d- and l-types, l-malic acid, the sole monomer of PMLA, exists as an intermediate metabolite in the tricarboxylic acid (TCA) cycle [3]. Because PMLA finally decomposes into the unique hydrolysates of l-malate, it possesses minimal immunogenicity or cytotoxicity while still maintaining full biodegradability and biocompatibility [4]. In addition, given the high density of carboxyl groups in its side chains (~ 862 free carboxylates per PMLA of a molecular weight Mw = 100 kDa) (Fig. 1), PMLA is highly water-soluble and chemically reactive, making it ideal for further pharmaceutical attachment.

Fig. 1
figure 1

Structures of PMLAs, including structures of α-, β-, or α, β-types of polymalic acid and malic acid in its l- or d-form

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Polymalic acid was first isolated from the secretions of Penicillium cyclopium in 1969 [5] and has been examined for its biochemical roles in protein homeostasis and nucleic acid synthesis [6, 7]. PMLAs were not chemically synthesized until benzyl malolactonate was polymerized to form PMLA of Mw < 6 kDa in 1979, thereby considered as a potential drug carrier for the first time [8]. With the rapid development in bioresource technology, research efforts on the bioproduction of PMLAs with relatively large Mw have intensified [1, 9]. Using economic fermentation, PMLA biosynthesis is taking steps toward a large-scale and continuous standardized production [9]. Lately, the search for polymalatase that is responsible for PMLA biosynthesis has shown exciting results, especially with the input of the CRISPR/Cas9 gene editing technique [32]. Moreover, PMLA bioproduction and cell growth were decoupled in Aureobasidium sp. when CuSO4 was added into the preparative cell culture (but not into the reaction mixture), possibly because CuSO4 initially triggered some enzymatic activity involved in the PMLA biosynthetic pathway, although its presence eventually decelerated cell growth [69].

As shown in Fig. 3, the addition of CaCO3 into basal medium serves as a switch from the intramitochondrial oxidative pathway of PMLA biosynthesis to the cytosolic reductive pathway, enhancing the fixation of CO2 by pyruvate carboxylase to continue malate production without involvement of the TCA cycle. Theoretically, for each molecule of glucose consumed, more malic acid could be generated in the reductive pathway than in the oxidative pathway; thus, carbonate also serves as a supporting carbon source of significant importance. In P. polycephalum, the optimal productivity of PMLA was achieved in the presence of CaCO3 when biomass was slightly increased and in this case the addition of intermediates of the TCA cycle (e.g., fumarate, succinate) did not contribute to enhanced productivity [70]. The buffering effect of carbonate not only promotes the availability of dissolved CO2 but also adjusts the acidity of the culture medium to prevent a sudden drop in pH that could undermine PMLA production. In A. pullulans, a variety of strains has been reported to produce PMLA with high yields in the presence of CaCO3, whereas the omission of CaCO3 in cell culture leads to an extracellular accumulation of other products, such as pullulan and lipids, rather than PMLA [11, 71, 72]. Intriguingly, replacement of CaCO3 with K2CO3 or Na2CO3 may either suppress the production of PMLA or shorten the polymeric length, accenting the additional functions of Ca2+ in two ways: (1) via the prompt precipitation of PMLA in the culture by the formation of Ca2+-PMLA, thereby evading hydrolase degradation; and (2) by possibly involving Ca2+ signaling in support of PMLA synthetase activity [47, 61, 73].

The optimized inoculation for PMLA bioproduction requires temperatures of 20–25 °C plus pH = 4.0 for A. pullulans strains [74] or temperatures of 24–28 °C plus pH = 5.5 for P. polycephalum [70]. Sufficient airing and appropriate stirring are crucial for the reactor settings [75]. The addition of a nonionic surfactant (e.g., Tween 80) into A. pullulans cultures at the cell growth stage can increase PMLA productivity by partially solubilizing the plasma membrane and upregulating a variety of metabolic enzymes involved in PMLA biosynthesis and cellular energetics [76]. PMLA producers from various marine and terrestrial habitats have been found and isolated for high-yielding strains [72, 77]. Simultaneously, enzymes governing in vivo malate production have been overexpressed by genetic modulation or exogenous stimulation to promote PMLA productivity, such as pyruvate carboxylase and malate synthase [78, 79]. In addition to optimizing the fermentation process, researchers have implemented new techniques, such as solid-state fermentation and membrane-assisted devices, to improve PMLA productivity and lower the production cost [80, 101]. Another is that the complexity and heterogeneity of the cancer microenvironment significantly prevent drug accumulation and action at the tumor site [102]. Given these problems, delivery systems that modify pharmacokinetics in favor of improved pharmacodynamics are greatly desired. Thanks to its excellent water solubility, total biodegradability, non-immunogenicity, and minimal toxicity at maximal dosage, PMLA has an unrivaled biological compatibility, serving as a competent platform for the further encapsulation and delivery of diagnostically and/or therapeutically active molecules to the target site. Moreover, the high chemical reactivity of its abundant pedant carboxylic acid groups guarantees PMLA’s exceptional loading capacity as well as its multifaceted functionality, providing a multipurpose carriage in order to maximize synergism and minimize side effects. In particular, for drug delivery to solid tumors, PMLA-based stimuli-responsive platforms at nanoscale are especially useful as they circumvent the unique features of the tumor microenvironment [3]. These PMLA platforms can enhance the permeability and retention of nanoparticles in irregular blood vessels, promote hypoxia- or redox-mediated drug targeting in the extracellular matrix, and exert a pH-triggered drug payload release in acidic tumor cells.

Stepwise conjugation through pendant carboxylic acids in PMLA

Through step-by-step chemical synthesis, PMLA extracted from P. polycephalum can be combined with different functional fragments (such as polyethylene glycol or PEG, antibodies, peptides, nucleotides, fluorescent tracers, or chemotherapeutic drugs) through direct or indirect (through spacer) conjugation to create a set of biopolymers named Polycefin [12, 103]. Among them, different pendant moieties along the PMLA backbone can be assigned with different biological missions; for instance, PEG can reduce nonspecific protein binding and prolong circulation time in the bloodstream, whereas a specific antibody can be designed to target the pairing receptor of target tumors and deliver a high concentration of drug [103]. Antibodies (such as the transferrin receptor monoclonal antibody, TfR mAb) or membrane-disruptive peptides (such as l-valine or trileucine) help penetrate biological barriers to protect nanoconjugates from early degradation and therefore increase the final drug accumulation at the destination; moreover, antisense oligonucleotides (AONs), synthetic DNA oligomers that can form highly sequence-specific hybrids with target RNA, are attached to inhibit the overexpressed laminin-8 in human glioblastoma multiforme (GBM) [104]. Finally, therapeutic synergism can be achieved as well as visually proven by bonding theranostic segments in Polycefin, including inhibitory nucleic acids, small-molecule chemical drugs, and multimodal imaging agents.

Using naturally derived PMLA as a nanoplatform, a series of Polycefin-based imaging and treatment substances was designed and fabricated with the goal of penetrating the blood–brain barrier (BBB) or blood–brain tumor barrier (BBTB) for diagnosis and therapy of neurological disorders, including primary and metastatic brain tumors and Alzheimer’s disease. Purified PMLA from the culture medium of P. polycephalum of Mw = 80 kDa with a polydispersity of 1.3 was covalently linked to gadolinium-DOTA (Gd-DOTA, a contrast enhancement tracer based on Gd) and curcumin [105]. These complexes, having a sub-10-nm size (8.6 nm), enabled the magnetic resonance imaging (MRI) detection of amyloidal beta (Aβ) plaques in ex vivo brain samples of human and mouse models of Alzheimer's disease [105]. Later, dual-imaging modes of the fluorescent dye Alexa 680 and the MRI reagent Gd-DOTA were added to a PMLA scaffold, and the complex was further conjugated with therapeutic monoclonal antibodies (trastuzumab for HER2 targeting and/or cetuximab for EGFR targeting) in order to easily differentiate a simultaneous diagnosis of HER2+ and EGFR+ brain tumors (Fig. 5) [106].

Fig. 5
figure 5

PMLA-based nanoparticles for tumor theranostics. a (i) MRI brain scans of mice with double tumors: a GBM (U87MG, EGFR+) in the left hemisphere and a metastatic breast cancer (BT-474, HER2+) in the right hemisphere after IV injection of (ii) targeted PMLA conjugated with Gd-DOTA/cetuximab/mouse TfR-mAb/Alexa 680, with (iii) quantitative analysis of MRI contrast in tumors and (iv) confirmation of MRI diagnosis by immunohistochemical analysis. Reprinted from ref. [106], with permission from the American Chemical Society, Copyright 2015. b (i) MRI scans of mice with double tumors, a primary GBM (U87MG, EGFR+) in the left hemisphere and metastatic breast cancer (BT-474, HER2+) in the right hemisphere after IV injection of (ii) targeted PMLA conjugated with Gd-DOTA/trastuzumab/mouse TfR-mAb/Alexa 680, with (iii) quantitative analysis of MRI contrast in tumors and (iv) confirmation of MRI diagnosis by immunohistochemical analysis. Reprinted from ref. [106], with permission from the American Chemical Society, Copyright 2015. c Schematic presentation of (i) PMLA-based nanodrugs, (ii) stereotactic implantation of brain tumors, and (iii) proposed mechanism of action. (iv) Kaplan–Meier animal survival curve for treatment of HER2+ BT-474 brain metastasis, EGFR+ A-549 brain metastasis, EGFR+ triple-negative MDA-MB-468 brain metastasis. The corresponding PMLA nanoconjugates improved survival by 57%, 66%, and by 114%, respectively (from left to right). Reprinted from ref. [106], with permission from the American Chemical Society, Copyright 2015

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Recently, by switching the navigator antibody in Polycefin, a BBB-penetrating peptide Angiopep-2 (AP-2) was chemically attached onto the PMLA base (Mw = 50 kDa), together with LLL to facilitate an endosome escape and rhodamine (rh) for fluorescent tracing [107]. Having a hydrodynamic diameter of < 5 nm, this new version of Polycefin (PMLA/LLL/Angiopep-2/rhodamine, i.e., P/LLL/AP2/rh), unlike either P/LLL/rh or P/AP2/rh that could not substantially penetrate BBB, showed a rapid accumulation in brain regions 30 min after i.v. administration and a fast clearance after 4 h (Fig. 6), demonstrating a promising neurological delivery platform [107]. Moreover, AP-2–guided PMLA conjugates with multiarm Gd contrast agents, which measured 15.8–20.5 nm in size, also showed a successful delivery to mouse glioma models and an enhanced brain tumor detection [108]. TfR- or Ap-2–directed PMLA in covalent conjugation with checkpoint inhibitors, including cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and programmed cell death 1 (PD-1) antibodies, was successfully delivered into brain tumor cells, triggering local immune responses and prolonging the survival of intracranial GBM-bearing mice [109]; similarly, chlorotoxin (CTX), which showed exceptional BBB-penetrating and specific GBM-binding ability, was conjugated to Polycefin together with a clinically approved near-infrared (NIR) fluorescent tracer, indocyanine green (ICG), making P/LLL/CTX/ICG nanoconjugates, and was injected i.v. into GBM-bearing mice for an NIR fluorescence-facilitated brain tumor resection, showing a high precision and the completion of tumor removal (Fig. 7) [110].

Fig. 6
figure 6

P/LLL/AP2/rh nanoconjugates for BBB penetration. a Chemical composition of P/LLL/AP2/rh, optical imaging data showing nanoconjugate permeation of the cerebral cortex (A1-3) and average nanoconjugate fluorescence in brain parenchyma (B1-3). b Pharmacokinetics of nanoconjugate in serum (P/LLL/AP2/rh and P/LLL/rh) and brain tissue (optical imaging of the sagittal sinus blood vessel). c Vascular fluorescence intensity profile (right) for the sagittal sinus vessel as indicated with a yellow line (only 30 min drug accumulation shown in the left). d Time dependence of nanoconjugate fluorescence intensity in brain parenchyma. Reprinted from ref. [107], with permission from the American Chemical Society, Copyright 2019

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Fig. 7
figure 7

P/LLL/CTX/ICG nanoconjugate for NIR-guided resection of brain tumor. a Composition of P/LLL/CTX/ICG and its injection into murine model of brain tumor. b Ex vivo fluorescence microscopy showing extravasation of P/LLL/CTX/ICG across BBB and intense distribution outside blood capillary and next to nuclei in tumor cells. c Biodistribution of P/LLL/CTX/ICG and d Real time NIR-imaging and surgical resection of GBM, 4 h after IV injection into mouse tail vein. Reprinted from ref. [110], with permission from Elsevier, Copyright 2019

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Similar to the design of Polycefin, many other PMLA-derived nanoconjugates have been developed for cancer targeting and treatment, most of which are based on the use of chemically synthetic polymers [111, 112]; for instance, one PMLA nanocomplex was constructed via stepwise chemistry: first, an amount of proton-sponge material polyethylenimine (PEI) was used to reverse the negative charge of PMLA, thus becoming positive; next, doxorubicin (DOX) and the transactivator of transcription (TAT) peptide were conjugated to the pendant carboxylic groups in PMLA through a pH-responsive linker and a primary amine-contained maleimide spacer, respectively, which formed PMLA-PEI-DOX-TAT micelles in aqueous solution; finally, six-armed PEG was conjugated to 2,3-dimethylmaleic anhydride (DMMA) or succinic anhydride (SA) and then added to solutions of PMLA-PEI-DOX-TAT micelles to dock on their outer surface through electrostatic adsorption, yielding PMLA-PEI-DOX-TAT@PEG-DMMA and PMLA-PEI-DOX-TAT@PEG-SA nanocomplexes (Fig. 8) [113]. PMLA-PEI-DOX-TAT@PEG-SA was applied as a non-charge-reversal control because of its structural similarity (but reverse pH-sensitivity) to PMLA-PEI-DOX-TAT@PEG-DMMA (Fig. 8b) [113]. After injection into the bloodstream, the shielding effect of PEG and the negative charge of the nanocomplexes at a physiological pH prevented 120-nm-sized particles from immediate recognition and clearance by the reticuloendothelial system, thereby improving their pharmacokinetics and allowing access into the acidic tumor microenvironment; DMMA (but not SA) was responsively disposed of, thus providing the positively-charged TAT-guided nanoparticles an advantage in penetrating tumor cells and unloading DOX drugs for tumor inhibition (Fig. 8c) [113].

Fig. 8
figure 8

PMLA-PEI-DOX-TAT@PEG-DMMA nanocomplex for anticancer activity. a Illustration of PMLA-PEI-DOX-TAT@PEG-DMMA prepared for effective drug release in cells. b Characterization of PMLA-PEI-DOX-TAT@PEG-DMMA. c The antitumor effect of nanocomplex on nude mice bearing A549 cells subcutaneously. Reprinted from ref. [113], with permission from Ivyspring International Publisher, Copyright 2017

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It is evident, therefore, that PMLA can serve as a flexible and versatile platform for further add-ons; nevertheless, these additions require a multi-step synthesis of different functional groups and the repetitive purification of intermediate products, resulting in a small yield of a final product potentially contaminated with unwanted side products. At the same time, the aqueous medium needs to be optimized for PMLA conjugation to prevent hydrolysis of the PMLA backbone, which would partially or completely negate functionalization. This optimization is especially challenging in the manufacturing industry and for biosafety assessments before clinical trials. Furthermore, through conjugation to form covalent bonds with pendant carboxylic acids, the chemical structures of the various attaching moieties might not remain intact, causing a possible loss of function to an unknown degree.

Simultaneous modification on pendant carboxylic acids in PMLA

Different from the stepwise conjugation described above, a simultaneous modification through chemical transformation or noncovalent binding of pendant carboxylic acid groups can be used to form PMLA-based nanostructures. With this method, after PMLAs are purified from fermented microorganisms, their malic acid units are transformed into partial blocks of opposite hydrophilicity (e.g., via benzylation) or are bound to electrolytes of opposite charge (via cationic complexation). The PMLA-based nanostructures obtained are thus restabilized as nanoscale platforms, enabling the loading and controlled release of pharmaceutical molecules.

Methylated PMLAs were obtained in a typical one-pot synthesis in which different ratios of diazomethane over PMLA (Mw = 34 kDa) in acetone were mixed for the reactions, and partial esterification of the pendant carboxylates was accomplished at random [94]. In aqueous solutions, the size of the newly formed polymer nanoparticle was determined to be between 3.0 and 5.2 nm; comparatively, from PMLA to a more methylated PMLA, the surface charge became less negative and the rate of hydrolytic degradation beginning with the breakage of the methylated carboxylate groups followed by hydrolysis of the PMLA backbone was reduced [94]. Hydrophobic drugs can be encapsulated within the methylated PMLA region through hydrophobic interactions, and their release occurs with the hydrolysis of the ester bonds, a process that is independent of cargo load but modifiable according to the hydrophobicity of copolymer [95]. This drug release can be further controlled or accelerated by the addition of certain external stimuli to the biological milieu, including changes in pH, ionic strength, redox, etc.

Polymalic acid under most biological conditions becomes a polyanion that possesses a number of carboxylate ions, which can easily interact with cationic moieties to form stable complexes [114]. In a typical case at pH 4.0, different moles of positively-charged DOX (pKa = 8.6) were added into aqueous solutions of PMLA with a fixed mole (in units of malic acid; i.e., DOX/PMLA = 0.1:1, 0.25:1, and 0.5:1). The increasing amount of DOX made the solution change from colloidal suspension to reddish precipitation, and > 90% added DOX formed ionic complexes with carboxylic acids of PMLA via their amines [114]. After DOX was added into the PMLA solution, this hydrophobic drug stayed in the inner core of the formed spheres, whereas the hydrophilic polymer constituted the outer surface through self-assembly (Fig. 9a). With more DOX partaking in the interaction, the attached drug itself formed π–π stacking (a strong cohesive force that significantly shrinks the inner core to make nanospheres) between their aromatic sheets [114]. The loading efficiency of DOX was shown to be almost three times higher than that using the physical encapsulation of DOX by methylated PMLA; in comparison, the drug released by the spherical PMLA/DOX ionic complexes was governed by both changes in pH and ionic strength, through a mechanism in which an acidic pH could quickly degrade PMLA’s outer surface in the spheres and, to a lesser extent, the increased concentration of electrolytes would diminish the ionic bonding between DOX and PMLA [114].

Fig. 9
figure 9

PMLA-based ionic complexation. a Schematic illustration of PMLA/DOX particle formation and drug loading and release. Reprinted from ref. [114], with permission from Elsevier, Copyright 2014. b Supramolecular structure of CnATMA-PMLA ionic complex. Reprinted from ref. [115], with permission from Wiley–VCH, Copyright 2007. c Formation of Cys-CS/PMLA particles and their pH-sensitive release of antibiotics to treat Helicobacter pylori in the stomach. Reprinted from ref. [117], with permission from Elsevier, Copyright 2018

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Alternatively, anionic PMLA can first react with cationic compounds to form a supramolecular assembly where its property of loading and releasing drugs becomes further adjustable by choosing a variety of cationic counterparts. In a pilot study, alkyltrimethylammonium (ATMA) surfactants with different lengths of alkyl chains (i.e., CnATMA, where n represents the number of carbon atoms in the alkyl chain) were chosen to form ionic complexes with PMLA (CnATMA-PMLA). The ionic complexes were studied to determine their interplay with antibiotics for drug loading and release [115]. Results showed that the ionized surfactants in the aqueous solutions were adsorbed by polyacids through electrostatic interaction, and, between the PMLA backbones, they were intercalated by hydrophobic surfactants with long alkyl chains, showing an ordered nanostructure with a paraffinic phase (Fig. 9b) [115]. For this reason, a higher degree of hydrophobicity, which is achieved with a longer alkyl chain in CnATMA, helps to prevent a water/enzyme attack that would break down the ester bonds in PMLA backbone, resulting in a slower hydrolytic or enzymatic degradation of CnATMA-PMLA and consequently releasing single units of erythromycin-bound malates at a slower rate (i.e., the degradation and erythromycin-releasing rate both followed the order PMLA > C14ATMA-PMLA > C18ATMA-PMLA) [115].

Polymalic acid of a high Mw (205.4 kDa) produced from the liquid fermentation of Aureobasidium pullulans var. pullulans MCW, was mixed with cysteine-conjugated chitosan (Cys-CS) via ionic gelation at different weight ratios to form particles of different sizes and surface charges [116]. Because of electrostatic association, positively charged Cys-CS/PMLA particles formed intact spheres at pH = 1.2 (simulating gastric acid), dramatically swelled at pH = 6.0 (simulating gastric mucosa), and completely dissociated at pH = 7.0 (simulating Helicobacter pylori). These particles were chosen to load antibacterial amoxicillin, further showing a pH-triggered release to inhibit the growth of Helicobacter pylori while leaving stomach cells unharmed (Fig. 9c) [117]. A strong intermolecular binding between thiolated Cys-CS/PMLA and glycosylated mucin was also confirmed, displaying an excellent mucoadhesive potential for enhanced delivery [116]. These Cys-CS/PMLA particles provide the scaffold for orally administered drugs designed to treat stomach bacterial infections. The subsequent inclusion of biosurfactants (rhamnolipids) or carbon dots into the CS/PMLA system showed a superior loading capacity over 70% with a particle size of ~ 200 nm and an antimicrobial effect against H. pylori of 99% [118, 119].

In addition, a biodegradable thin film was prepared after a layer-by-layer assembly of PMLA and chitosan, revealing an excellent capacity for the controlled release of proteins, including lysozyme and basic fibroblast growth factor (bFGF) [120]. To incorporate proteins, a tetralayer architecture (of chitosan/PMLA/protein/PMLA)n was assembled, having a thickness of 69.2 nm and 10.7 nm for lysozyme and bFGF films, respectively (The bFGF film per tetralayer was significantly thinner than the lysozyme film because of the more potent nature of bFGF; therefore much less protein was able to be applied to the film), and their film growth exhibited a high degree of linearity between film thickness and the number of tetralayers. This tetralayer architecture demonstrated an exceptional diffusiveness between the layers of film, which would be advantageous for cargo release [120]. It was shown that the chitosan addition to the (chitosan/PMLA/protein/PMLA)n tetralayer prevented the abrupt release of biologics in the case of (protein/PMLA)n bilayers, sustaining a timed release due to the hydrolytic degradation of PMLA and the accompanied dissociation of ionic complexes in response to changes in environmental pH and ionic strength; comparatively, dissembled chitosan could preserve the activity of the released proteins by preventing them from being denatured or proteolyzed [120].

In PMLA-based ionic complexation, whether PMLA forms stable complexes directly with positively-charged functional compounds of low Mw (e.g., small-molecule drugs) or first interacts with oppositely charged polyelectrolytes of high Mw and then with functional compounds, the complexes undergo a completion of reactions in aqueous solutions. These series of reactions aid in unleashing more absorbed therapeutic or diagnostic reagents with the addition of stimuli or by stimuli-triggered self-degradation of PMLA main chain compared with free drug diffusion. Accordingly, PMLA hydrolysis, together with the dissociation of the ionic complex, liberates the drug-bound malate units.

Conclusion and perspective

Here we review the recent advances in the chemical synthesis and bioproduction of PMLAs, their physicochemical and physiochemical characteristics, their biomedical applications as delivery platforms for disease diagnosis and treatment, and the unique chemical methods used to engineer PMLA-based nanomaterials. Produced from biological sources rather than chemical reactions, PMLA has several advantages as a unique platform for theranostic delivery in the clinical translation. Firstly, manufactured through fermentation engineering of microorganisms, PMLA is a biorenewable natural product with promising full-scale industrialization. Secondly, water solubility of PMLA is unrivaled when compared with water-insoluble biopolymers, such as cellulose, polyhydroxybutyrate, etc., or biopolymers that are only water soluble in salt form, such as polyglutamic acid. More importantly, this excellent water solubility is inherited by many PMLA-derived complexes, maintaining their suitability as delivery platforms. Thirdly, pendant carboxylic acids along the main chain of PMLA offer several opportunities for further covalent conjugation or ionic complexation, making PMLA a resourceful carrier for therapeutic and diagnostic delivery. Fourthly, the release mechanism of a PMLA carrier is mostly due to the hydrolytic breakage of the ester bonds in its backbone, although it could be complemented with the dissociation of other intermolecular forces, such as ionic bonds. The timing of this release profile greatly avoids a premature cargo leakage before delivery to the diseased lesion. The degradative or hydrolytic rate of PMLA under physiological conditions could be favored by pharmacokinetics. Lastly, the full biodegradability can make PMLA adaptable to many formulations, including orally, by injection, or even through implantable medical devices, as the final degradation products comprise of sole l-malates. These products can be further utilized in the TCA cycle and metabolized into CO2 and water, constituting no secondary harm to the body or no residual deposition in the body.

However, after decades of PMLA research and technological development, challenges still remain. The first problem lies in the PMLA’s unscalable production and functionalization for medicinal purposes. Following current manufacturing flow and purification processes, the regular yield of bioproduced PMLA remains on a scale of grams per batch, taking up to two weeks per production cycle [59]. Further functionalization that readies PMLA-based compounds for biological use, especially through conjugation chemistry, only produces the final product on a scale of milligrams after purification steps; moreover, each purification step may require different solvents, apparatuses, reaction parameters, etc., all leading to complications in scalability from bench to production plant, let alone from clinical trial to bedside [60].

The second challenge remains that the genes responsible for PMLA production in several microorganisms are yet to be fully uncovered. Once identified, these genes could be introduced into the commonly used industrial microbes for the biosynthesis of this precious natural product using metabolic engineering, thereby largely lowering the manufacture cost, boosting the yield, and commercializing the product. For this reason, the Mw of PMLA cannot be controlled during its biosynthesis, so it is impossible to tailor the length of bioproduced PMLA as desired. In parallel, the relationship between the Mw of PMLA and its suitability as a delivery platform also needs to be determined. The higher the Mw of PMLA, the greater its loading capacity and the longer its delivery time is before its hydrolytic degradation; however, this relationship has not been quantitatively defined or precisely assessed.

Last but not least, there are unexplored opportunities in which PMLA could be used for more applications other than a delivery platform. With unrivaled hydrophilicity, PMLA-based drug delivery can be applied to many fields insofar uncharted, such as kidney diseases and urological cancers/infections. Besides, as a fully degradable biopolymer, PMLA could be useful in fields other than biomedical research, such as the food or pharmaceutical industry for external packaging. Its applicability to these fields would only help promote research into its medicinal development.

In closing, PMLA is currently one of remarkable materials for many biomedical applications, including pharmaceutical delivery and tissue engineering. Its nontoxic, non-immunogenic, and fully degradable features make it a perfect medicinal platform, holding great potential in targeted biomolecular delivery and many other pharmaceutical and biological applications.