Supercritical carbon dioxide design strategies: from drug carriers to soft killers

1. Introduction

In the late 1980s, chemists started to be more aware of the environmental problems caused by an excessive production of toxic wastes. The formalization of the principles of green chemistry [1] and green engineering [2], pioneered by Anastas and co-workers, are conceptual milestones to the sustainability awareness and were later condensed by the group of Poliakoff in a very simple way for easy dissemination [3].

The use of supercritical fluids is considered as an important alternative strategy within green chemistry to replace volatile organic compounds, thus empowering novel clean technologies [4]. A major research focus has been the investigation of processes using supercritical carbon dioxide (scCO₂) to replace or minimize the use of traditional solvents. Particularly concerning the polymer research area, scCO₂ has been widely investigated [5–11] showing a great potential in providing some key advantages for polymer synthesis and polymer processing when compared to conventional methods. The most evident features are their pressure tuneable physical properties and solvent power. The ability to rapidly vary the solvent power of dissolved compounds, and thereby the rate of supersaturation and nucleation, is crucial for particle formation steps. Furthermore, for pharmaceutical applications, scCO₂ is an ideal medium because it has a relatively mild critical temperature, it is non-toxic, non-flammable and leaves the system with no residues just by decompression [12]. Although the significant costs associated with polymers processing using high-pressure add significant value to the final products [13], this type of technology constitutes a greener approach since organic solvents are avoided.

Challenges concerning the preparation of delivery systems loaded with clinically relevant quantities of therapeutic agents [14,15], functionalized with high-sensitivity and high-specificity probes for cancer targeting and imaging [16,17] and produced in a sustainable way, have been grabbed by multidisciplinary teams. The past few decades have witnessed a rise in the development of new therapeutics [18,19] which have been fostering the design of delivery platforms to treat a broad spectrum of diseases that were untreatable a decade ago [20]. Chemical and material scientists, pharmacologists, medical doctors and bioengineers have been involved to support the medical revolution that we are witnessing. In particular, the chemist’s contribution is crucial to develop and evaluate the technological process and bring key medicines with minimal environmental impact. The pharmaceutical manufacturing continues to involve an extensive use of organic solvents as reaction media in the synthesis of drugs, or as extracting agents for selectively isolating drugs from solid matrices or as anti-solvents for recrystallizing drugs from solutions. In the 1980s, therapeutic monoclonal antibodies and antibody-related products became the dominant product class within the pharmaceutical market [21]. The need for cost-effective supply of large quantities of these products put considerable pressure on the development of more efficient and sustainable antibody purification processes. Therefore, new affinity platforms inspired by the green chemistry principles are now coming to the scene [22–24].

The understanding of how the immune system behaves and how infectious diseases, inflammatory disorders, neurological affections and different types of cancer progress has enabled the development of pharmacotherapies that offer true benefits to the patients as opposed to simply treat the symptoms [25,26]. Major effective solutions coming to the market involve the delivery of biopharmaceuticals, including vaccines and therapeutic antibodies [27]. However, these biologics need to be injected directly to the blood stream to avoid structural degradation or decomposition during the digestion process [28]. Over the last years, a great effort has been made to investigate different non-invasive drug delivery systems (DDSs), namely the oral, sublingual, rectal, nasal, pulmonary and transdermal routes. These attempts led to a variety of formulation strategies to circumvent the obstacles linked to the non-invasive routes of administration. For instance, it has been shown that the nasal and the pulmonary delivery routes enable much higher bioavailability than the oral route for different drugs, due to the highly vascularized lung epithelium and the avoidance of first-pass hepatic elimination, gut wall metabolism and/or destruction in the gastro-intestinal tract [29].

Different formulation strategies have been considered when designing a DDS for a specific route of administration. For instance, while a dry powder formulation or nano- or micro-droplets are necessary for the pulmonary or nasal route via aerosolization or nebulization, respectively, the sublingual route requires the use of oral dispersive films as vehicles for the bioactive ingredient or macromolecule. The envisaged three-dimensional constructs, whether nano-/micro-sized particles, films, membranes or scaffolds imply the assembling of nanocarriers previously developed and their encapsulation or immobilization in the ultimate three-dimensionally shaped DDS. Recent reviews highlighted the potential of scCO₂ technology to assemble nano- to micro-size particles [30–34], to produce membranes with different morphologies [35] or scaffolds for drug delivery and other biomedical applications [36,37].

When administrating therapeutic agents via routes of administration that involve the use of devices such as needles, implants, inhalers or vaginal applicators, new health problems arise due to biofilms and device-associated infections [38]. Medical device infections are typically related with microbe colonization in devices and this contamination mostly happens by inoculation with only a few microorganisms from the consumer’s skin or mucous during use. Microorganisms commonly attach to living and non-living surfaces and form biofilms. Once adhered to the surface, microorganisms multiply and accumulate in multilayered cell clusters, which require intercellular adhesion, chemical interactions or quorum-sensing mechanisms [39]. In this state, microorganisms are persistently bound and are extremely resistant to antimicrobial treatment. Thus, research has focused on developing self-disinfecting surfaces that prevent the colonization of microorganism in medical devices, where scCO₂ has been used as a green technology [40].

Different research outputs have been reported concerning the development of DDSs for different routes of administration using supercritical fluid technology. The role of scCO₂ varies from reaction media for synthesis of oligo- or polymeric material for dug conjugation [41,42], solvent for impregnation of active agents into nano- to macro-sized devices [43,44] and also for processing of polymers [33,45–47]. This mini-review intends to present an up-to-date view of the role of scCO₂ on three main applications: (i) the synthesis of selected polymers with enhanced properties for drug and biologics conjugation; (ii) on the strategies to assemble target molecules and to functionalize molecules and surfaces; and (iii) development of antimicrobial agents. Special emphasis will be put on assembling active devices for the controlled release of drugs or biopharmaceuticals with the ultimate desired properties for drug delivery and also on the development of antimicrobial active devices. The underlined examples are analysed highlighting the pros and cons of the scCO₂-assisted technologies.

2. Drug and active delivery vehicles

When developing a drug formulation, many parameters must be optimized to enhance the drug performance. The drug vehicle design must be ideally adjusted considering solubility, degradation and renal clearance issues, but very often it is also intended to confer targeting or imaging abilities. For example, the vehicle might act not only as a therapeutic reservoir enhancing the drug solubility but also be able to guide and control the specific time-release of the drug, directly influencing its biodistribution and pharmacokinetics. In the case of a biologic DDS, specific features arise as the active macromolecule might lose its activity in consequence of environmental conditions during processing, storage or in the body. The targeted delivery of drugs to specific sites of action is being performed through two principal release mechanisms: polymer drug conjugates and nano-enabled delivery systems, or even combination of both approaches. Poly(ethylene glycol) (PEG) is still the standard polymer either for drug conjugation or for masking nanoparticles from clearance by kidney filtration [48]. Undergoing investigation on DDSs is focused on micelles, gold nanoparticles, magnetic nanoparticles, dendrimers, liposomes and the combined systems include polymer-coated nanoparticles. To date, only conventional methods have been used to prepare the drug products comprising liposome or polymer-drug-conjugated delivery systems already available on the market or under clinical trials. However, the conventional processes yield final products that might be contaminated to some extent with residual organic solvent and are also difficult to maintain scaled-up production. The polymer-based DDSs that have been brought to the market up to now contain PEGylated drugs (PEG-functionalized products). The increasing use of PEGylated products has brought to the scene some unfavourable effects: (i) non-specific interactions with blood can cause hypersensitivity reactions [49,50], (ii) accelerated blood clearance phenomena leading to changes in the pharmacokinetic behaviour of the drug; (iii) non-biodegradability; and (iv) toxicity of side products formed during PEG synthesis. These PEG drawbacks intensified the search for PEG alternatives. A variety of natural and synthetic polymers have been investigated but only the synthetic polymers which were produced in scCO₂ will be considered here. Polymers synthesized in scCO₂ present many advantages for biomedical applications over conventional processes due to the easy elimination of trace contaminants rendering highly pure materials. Oxazoline-based polymers (POxs) are the most likely substitutes to PEG [51] and have already been successfully synthesized in scCO₂ by cationic ring-opening polymerization (CROP) [52]. Table 1 compiles promising alternatives to PEG that have been proposed but limited to polymeric systems synthesized in scCO₂.

(a) Oxazoline-based polymers

Among the alternative polymers for drug conjugation, oxazoline-based polymers are recognized as the most promising PEG substitutes, in particular, poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) because of their demonstrated low toxicity and immunogenicity [58]. POxs are obtained through a CROP. A challenging but also versatile type of polymerization which allows the tuning of final polymer properties (such as hydrophilic, hydrophobic, fluorophilic) as it enables copolymerization with a variety of monomers and polymer chain end-capping. When the reaction is performed in scCO₂, oligo-oxazolines (OOxs) are obtained [52]. The schematic synthesis of OOxs and their structure are shown in figure 1a. The structure of PEG is also shown for comparison (figure 1b).

• Download figure
• Open in new tab
• Download powerPoint

The synthesis of these polymers using conventional solvents [59], microwave-assisted [60,61] or green scCO₂-assisted methodologies [52] has been previously described. Table 2 displays the advantages and disadvantages of scCO₂ polymerization in comparison to the other methods.

From table 2, we can easily conclude that the supercritical CO₂ route is not only a viable alternative, but brings also additional advantages to the polymerization process. A particular feature observed for OOxs synthesized in scCO₂ is their intrinsic blue fluorescence, which is achieved by a carbamic acid insertion at the oligomer starting end during polymerization (figure 2). This is contrary to the common assumption that CO₂ was inert in this type of polymerization [5].

• Download figure
• Open in new tab
• Download powerPoint

No matter which route is followed, reported results demonstrate that OOxs and POxs can be prepared with different architectures and end-capped with selected functional groups for adequate conjugation [62].

(b) Polyurea dendrimers

Dendrimers have been actively studied as carriers for targeted delivery of small-molecule drugs but their translation into clinics is very limited [65]. Only two examples are under clinical evaluation: OcuSeals (Beaver-Visitec International) for ocular administration, which is a hydrogel-dendrimer liquid ocular bandage [66] and VivaGel (Starpharma Holdings Ltd), a topic gel for vaginal application composed of a poly(lysine) dendrimer decorated on surface with 32 naphthalene disulfonate units (http://www.starpharma.com/vivagel/vivagel_clinical_trials, accessed on 23 May 2015). The first synthesis of dendrimer polymers in scCO₂ was recently reported by Restani et al. [67]. Taking advantage of using carbon dioxide as a solvent and as a reagent (C1 feedstock), polyurea (PURE-type) dendrimers were synthesized by reacting tris(2-aminoethyl)amine with carbon dioxide (figure 3a).

• Download figure
• Open in new tab
• Download powerPoint

Importantly, the incorporation of CO₂ in the dendrimer backbone produced a blue fluorescent polymer, which intensity was found to be pH dependent. Moreover, PURE-type dendrimers were found to be water soluble and biocompatible, showing no cytotoxicity even at high concentrations, the major drawback of reported dendrimers (e.g. PAMAM-type). Moreover, PURE dendrimers were found to form dendriplexes with siRNA (PURE_G4-siRNA), be able to perform its deliver to cells with high transfection efficacy and efficiently shutdown the expression of a specific gene [56]. Also, the PURE_G4-siRNA dendriplex dramatically decline cell oxidative stress and cytotoxicity of the carrier system even at high concentrations when compared with PURE_G4.

(c) POxylated drug delivery systems

Considering the high potential of poly(2-oxazolines) (POx) as PEG alternatives, several studies were reported in the literature bearing in mind drug carrier applications [48]. Micelles of PLA-PEtOx-PLA (PLA: poly(lactic acid)) were prepared for doxorubicin delivery and paclitaxel loaded micelles of PEtOx-poly(ϵ-caprolactone) were shown to possess high efficiency as a paclitaxel release system. Apart from water-soluble and biocompatible, copolymers of 2-oxazolines were shown to possess several important properties required for drug conjugation and also for in vivo gene delivery including (i) easy conjugation to proteins [68], (ii) the ability to stabilize particles against salt-induced aggregation, (iii) the ability to resist extracellular polyplex unpackaging, (iv) the potential to be degraded into non-toxic components after cellular uptake, and (v) efficient delivery of plasmid to cultured cells [69]. The reported studies encompassed 2-oxazoline-based materials synthesized using conventional methods. Recently, Restani et al. [57] demonstrated for the first time that oligo-oxazolines synthesized in scCO₂ could be conjugated to improve the performance of drug nanocarriers. Following a POxylation approach, polyurea dendrimers were complexed with living oligo-oxazolines synthesized in scCO₂ (figure 3b). The synthesized PURE_G4-OOxs dendrimers are water soluble, pH-responsive and present intrinsic fluorescence. These water-soluble core–shell nanocarriers were found to be less toxic than pristine PURE_G4 dendrimer in terms of cell viability and absence of glutathione depletion, two of the major side effect limitations of current therapeutic polymers. Using scCO₂ technology, the authors were able to load the nanocarriers with paclitaxel. Remarkably, the authors found a 100-fold reduction of paclitaxel IC₅₀, relatively to the free drug, in the tested formulations. PURE_G4-OOxs were also found to act as good transfection agents, through a mechanism involving an endosomal pathway. These results show that supercritical-assisted polymerization is a powerful tool to prepare efficient DDSs opening new trends to low-cost and highly efficient chemotherapeutics.

3. Antimicrobial strategies

Microbial infections in humans and materials represent a critical health problem and economical challenge in modern society, aggravated by the appearance of new antibiotic-resistant bacterial strains. Several approaches have been pointed out in the last years to control and ideally eradicate this problem, namely trough chemical disinfection or through the use of materials with antimicrobial properties [70] (http://www.starpharma.com/vivagel/vivagel_clinical_trials, accessed on 23 May 2015). The most common solution, chemical disinfection, is usually accomplished by the use of disinfectants that ultimately lead to environmental pollution and could also trigger the development of bacterial resistant strains. Antimicrobial polymers have been considered a new class of disinfectants, which have been recognized even as alternative antibiotics. The first polymer disinfectant that became commercially available was poly(hexamethylene biguanidinium hydrochloride), thoroughly investigated by Ikeda et al. [71]. Antimicrobial polymers widen up a whole new range of therapeutic solutions enabling the tuning of different properties by combining different polymer backbones and functionalities [72]. Interestingly, antimicrobial polymers can be used to modify surfaces, without losing their biological activity, which enables the design of surfaces that kill microbes without releasing biocides, solving numerous contaminations mainly in healthcare facilities [73]. In 1965, Cornell & Dunraruma reported, for the first time, antimicrobial polymers and copolymers based on 2-methacryloxytroponones [74]. Since then, different polymeric structures with antimicrobial properties have been reported in literature and the number of publications in this topic per year is growing fast. Just in the last decade, according to Web of Science, the number of publications has tripled.

Several studies have been reported towards the development of an ideal antimicrobial polymer. For that, some basic requirements should be taken in consideration: (i) easy and inexpensive synthesis; (ii) stability during usage and storage at the conditions of its intended application, do not lead to emission or generation of toxic products; (iii) biocompatibility; (iv) easy recovery of activity, if lost; and (v) active against a broad spectrum of pathogenic microorganisms in brief times of killing.

Known antimicrobial polymers are commonly classified in three distinct groups depending on the mechanism of action of each one [70] (figure 4).

• Download figure
• Open in new tab
• Download powerPoint

Polymeric biocides are expected to be less active than their corresponding analogues to steric hindrance, while biocide-releasing polymers could trigger the development of bacterial resistance and/or contamination of the surroundings. By contrast, biocidal polymers represent a good solution for the achievement of the basic requirements for an ideal antimicrobial polymer [75]. Interestingly, as bacterial cells carry a negative surface charge (due to the presence of membrane proteins, teichoic acids in Gram-positive bacteria and negatively charged phospholipids at the outer membrane in Gram-negative bacteria), positively charged polymers can be attracted to bacterial cell membrane and take their action [76]. For this reason, in the last decades, many efforts on the development of biocidal polymers relied on the use of antimicrobial polycations with quaternary ammonium and phosphonium [77]. Table 3 highlights the common scCO₂-assisted methods used to synthesize antimicrobial agents and to confer antimicrobial properties to different materials. In the subsequent sub-chapters, we will focus in the sustainable synthesis of biocidal polymers in scCO₂ and its importance in the development of antimicrobial POxylated surfaces.

(a) Biocidal oxazoline-based polymers

Polymeric quaternary ammonium salts can be prepared using different approaches that will result in distinct polymeric architectures, exhibiting diverse antimicrobial activities and potential applications [77]. Interestingly, polymers bearing ammonium salts are cationic and their mechanism of action is believed to depend on the fundamental characteristic of negatively charged microbial cell membrane. This aspect suggests that the development of strains resistant to this class of compounds is impaired, as it would require bacteria to change membrane structure [77].

Oxazoline-based polymers (POXs) are an attractive class of compounds due to their biocompatibility and synthetic versatility [65] and represent a unique approach to the development of new antibiotics. Recently, they have been explored as strong candidates for the development of new polymer therapeutics [91–94]. Waschinski et al. reported the synthesis of a series of oxazoline-based polymers, terminated with quaternary ammonium groups, prepared via standard CROP. The antimicrobial polymers activity was evaluated by determining the minimal inhibitory concentration against Staphylococcus aureus. Only poly(2-oxazoline)-based polymers containing alkyl ammonium functions with alkyl chains of 12 carbon atoms or longer were found to be active [93]. Correia et al. demonstrated that the synthesis of antimicrobial 2-substituted-2-oxazolines polymers can be successfully performed in scCO₂ in a more sustainable way. Several OOxs were synthesized by living CROP and later end-capped with a tertiary amine to produce quaternary-ammonium-terminated materials (OOXs-DDA). The synthesis and quaternization of branched oligo(2-bisoxazoline) was reported for the first time as well as its antimicrobial activity and cytotoxic effect, together with oligo(hydrochloride-quaternized ethylenimine), a polymer biocide, which was also synthesized by hydrolysis of a oligo(2-oxazoline) [90]. Table 4 summarizes the reaction conditions used for the synthesis of POxs-DDA using either the conventional or the scCO₂-assisted methods. Comparing the syntheses (conventional versus scCO₂), it can be concluded that, following the supercritical route, a more sustainable protocol was achieved with a clear reduction of purification steps, solvents and time (table 5).

(b) Bioactive POxylated surfaces

Antimicrobial polymers can be effectively designed to improve the materials properties. The possibility to incorporate, combine or graft such versatile polymers (with tuneable chemical and mechanical properties) into a surface broadens up the design of biocidal platforms. Antimicrobial surfaces can act by two ways: (i) a biopassive surface coating or (ii) an active bioactive-surface. The biopassive surface does not interact and kill bacteria but reduces the adsorption of proteins and, consequently, the adhesion of bacteria. This approach is mainly reported in the literature through the incorporation of hydrophilic polymers into surfaces, e.g. hydrogel coatings (mostly based on PEG) [95]. By contrast, bioactive surface coatings can kill bacteria on contact, either after the release of the biocide from the surface to the surroundings or after mere contact with the surface [96].

Non-fouling PMeOx-based coatings have emerged as a promising alternative for the replacement of PEGs by more biocompatible analogues [97]. Essentially, PMeOx-modified substrate is a biopassive approach to reduce biofouling and improve the ability to resist protein and bacterial adhesion (without killing) by enhancing materials hydrophilicity.

Importantly, bioactive materials that prevent biofouling should contain antimicrobial moieties attached onto polymeric backbones by covalent interactions to boost stability. Previous studies report the use of antimicrobial groups usually grafted to the polymer backbone or inserted in the main chain of polymers during synthesis by using monomers bearing antimicrobial moieties. Many studies also show that biocidal end-capping is an effective strategy [98]. However, the number of chemical steps and the use of organic solvents are not attractive from a sustainable point of view.

Recently, our group reported a green methodology to produce highly biocidal and biocompatible materials comprising antimicrobial oligomers in a chitosan (CHT) blend. The CHT biopolymer was chosen since it is a suitable three-dimensional support for application in tissue engineering [99] and controlled drug delivery [100], showing biocompatibility and biodegradability properties. CHT-based three-dimensional structures might represent an efficient alternative to conventional strategies to prevent protein adsorption and subsequent bioadhesion. For this work, cationic antimicrobial oligomers (oligo(2-methyl-2-oxazoline) quaternized with N,N-dimethyldodecylamine, oligo(2-bisoxazoline) quaternized with N,N-dimethyldodecylamine and linear oligoethylenimine hydrochloride), were incorporated into three-dimensional structures. By varying the antimicrobial polymer concentration in the casting solution, the hydrophilicity, permeability, BSA adhesion profiles and bacterial killing activity of CHT-based scaffolds could be tuned. Scaffold containing 20% (w/w) of oligo(2-bisoxazoline) quaternized with N,N-dimethyldodecylamine in its composition was the three-dimensional structure with best performance in repelling protein and killing Escherichia coli upon contact (1 mg of scaffold killed 99.9% of an initial E. coli inoculum of 5×10⁵ CFU ml⁻¹) [101]. Regrettably, the physical immobilization of OOxs-DDA in the blend impaired their activity against S. aureus cells as the antimicrobial oligomer cannot diffuse through the thicker peptidoglycan layer to be effective in the inner membrane. To surpass this limitation, attempts to immobilize OOxs-DDA at the surface scaffolds are currently under study.

4. Conclusion and future prospects

The studies reported show that polymers synthesized in scCO₂ namely OOxs, PURE-type dendrimers and their combined structures are powerful systems for applications in drug and gene delivery. The supercritical synthetic route for oxazoline-based polymers enables the production of intrinsically fluorescent polymers, the easy control of molecular weight, the end-cap of living polymers with different functional groups or their grafting to other materials.

Rapid advances in scCO₂-assisted methods have shown that viable alternatives to conventional approaches are affordable not only for polymer synthesis but also for functionalization and assembling of nano-DDS, rendering pharmaceutical grade products.

Authors' contributions

The manuscript was drafted by A.A.R. with input from V.D.B.B., T.C. and V.G.C. The work was coordinated by A.A.R. and the manuscript was finally revised by all authors. All authors gave final approval for publication.

Competing interests

We declare we have no competing interests.

Funding

The authors thank the financial support from Fundação para a Ciência e Tecnologia (FCT—Lisbon) and COMPETE through Contracts UID/QUI/50006/2013, IF/00915/2014 (T.C.) and SFRH/BD/74730/2010 (V.G.C.).

Footnotes

One contribution of 12 to a discussion meeting issue ‘Supercritical fluids: green solvents for green chemistry?’