ALEJANDRO SOSNIK
5.3 Temperature-sensitive Polymeric Micelles
5.3.1 Poly(ethylene Oxide)-Poly(propylene Oxide) and Other Polyether Amphiphiles
PEO-PPO block copolymers are the most extensively investigated micelle- forming copolymers.62–64One of the most appealing features of PEO-PPO water solutions is that they gel upon heating around 371C and they can be used to develop a variety of injectable systems for different biomedical applications.65,66 Based on their molecular structure, PEO-PPOs are classified into two groups:
(i) linear and bifunctional PEO-PPO-PEO triblocks (poloxamers, Pluronics, Scheme 5.1A) and (ii) X-shaped tetrafunctional derivatives (poloxamine, Tetronics, Scheme 5.1B). The regular derivatives combine terminal hydrophilic
CH3
CH3 CH3
CH3 CH3
A
B
CO-[NH-CH-(CH3)2] (-CH2-CH-)n
C
Scheme 5.1 General molecular structure of (A) poloxamer, (B) poloxamine and (C) poly(N-isopropylacrylamide).
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PEO blocks and central PPO ones and form micelles in aqueous medium, being the most relevant as pharmaceuticals. Conversely, there exist reverse-sequential counterparts that display terminal hydrophobic blocks and central hydrophilic ones. In less polar solvents, these derivatives generate reverse micelles that display a hydrophilic core and a hydrophobic corona. PEO-PPOs are biocom- patible for topical, oral and parenteral administration67 and, in general, they have shown good cytocompatibility.68–71 PEO-PPOs are not biodegradable, though molecules displaying molecular weights below 10–15 kDa can be bio- eliminated by renal filtration.72–74In addition, polymeric micelles with coronas made of PEO are sterically stabilized (Stealths) and minimize opsonization and uptake by macrophages, prolonging circulation timein vivo.75
Poloxamers and poloxamines are commercially available in different molecular weights and EO/PO molar ratios and some linear derivatives were approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as pharmaceutical excipients in medicines and medical devices.76–78The branched counterparts display two main distinctive features. An ethylenediamine central moiety (and two tertiary amines) that confers the molecule responsiveness to pH79–81 and enables chemical modification of the core.39,82For example, quaternization of poloxamines by means of N-alkylation not only partially suppressed pH-responsiveness, but it also modified the self-aggregation pattern (that resembled poloxamines at low pH), the drug-core affinity and the cytotoxicity.39,83,84 Even though poloxamines are dually responsive molecules to temperature and pH, they are discussed in this section because the stimulus usually exploited is temperature. However, changes in the aggregation/gelation/drug release under different pH conditions have been reported.85Poloxamines display two pKa values at 3.8–4.0 and 8.0 with minimal changes among derivatives of different molecular weight and HLB.86 At low pH both amine groups are protonated, coulombic repulsion prevents micellization, and CMT is shifted to a greater temperature.87At neutral pH, aggregation increases due to the partial disprotonation of the central group, becoming maximal at pH410–12 where molecules are completely unprotonated.87,88 The higher the pH is the greater the aggregation number, the larger the size and the more homogeneous the size distribution of poloxamine micelles.80 Accordingly, poloxamines display maximum solubilization capacity at pH48–10. In this framework, the release in biological microenvironments displaying reduced pH could be envisioned.
The mechanism behind the micellization of PEO-PPO and other amphiphilic copolymers is entropy-driven and mainly related to the release of water hydration molecules from the PPO blocks.89 Thus, the CMC and the CMT depend on the molecular weight and the EO/PO ratio; the greater the HLB and the lower the molecular weight, the higher the CMC and the CMT are.90 In addition, derivatives of greater molecular weight (and similar HLB) display smaller CMC and CMT.
Since PEO-PPOs are thermo-responsive, the CMC, the micellar size and the size distribution are also strongly dependent on the temperature. At higher
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temperature, the micellization tendency increases, leading to a smaller CMC and a greater fraction of molecules in micellar form.91–96 Consequently, the drug encapsulation and solubilization capacity of these copolymers grows with the temperature. Even though PEO-PPOs are non-ionic surfactants, the micellization process can also be affected by the presence of salts and different types of ions that produce salting-out or salting-in phenomena. In general, the greater the concentration of neutral salts, the smaller the CMC and the CMT.97–100In this context, some authors proposed a new parameter, namely the critical micelle salt concentration (CMSC), defined as the salt concentration at which micelles begin to form, though this parameter should be determined for every salt type. However, the analysis is not straightforward because extensive studies with different salts suggested that the behavior is not predictable based on the prior art.101,102
Most of the research at the interface of PEO-PPOs and drug encapsulation focused on poloxamers.64 It is worth noting that these developments were intended for a broad spectrum of administration routes, from topical to intravenous. In general, the addition of more amphile molecules above the CMC results in the formation of additional micelles and in the growth of the encapsulation capacity of the micellar system. Considering the relatively high aqueous solubility of PEO-PPOs, concentrations as high as 10–15% can often be obtained. In addition, the use of concentrations above 20–25% enables the generation of physical gels where the drug is primarily encapsulated within the micelles that form 3D networks. This is the reason that Pluronics F127 has become probably the most extensively investigated of all the poloxamers, a selection that is further supported by the fact that this derivative is currently FDA-approved for use in pharmaceutical products.64
Drug-loaded poloxamer micelles were also combined with physical means such as ultrasound to target the release of doxorubicin to tumors.103–109 Systems accumulated preferentially in the tumor by the EPR effect and then irradiation improved the cellular uptake of the drug. Pittet al.109suggested that ultrasound transiently destroys the micelles by cavitation, increasing the drug release in the irradiated area. Ultrasound-triggered release from micelles is tackled in Chapter 6.
In the last years, a few groups investigated more comprehensively the self- aggregation and the capacity of poloxamines to solubilize, stabilize physico- chemically and release different drugs.31,32,39,64,84,110–112
Alvarez-Lorenzo et al.113 assessed the encapsulation of the antifungal griseofulvin in 10%
solutions of poloxamine T904 under different pH conditions. The solubility increased three and six times at acid and alkaline pH-values, respectively.
A similar behavior has been shown for triclosan in poloxamine T1107 micelles, though solubilization was improved from 2mg/mL to up to 30 mg/mL (more than 15,000 times).31 However, this system was more complex owing to the ionization of the drug at pH410. The most relevant outcome was that triclosan-loaded micelles showed better antibacterial activity than the free drug, even against hospital resistant strains such as methicillin-resistant Staphy- lococcus aureus and vancomycin-resistant Enterococcus faecalis and in a Temperature- and pH-sensitive Polymeric Micelles for Drug Encapsulation 123
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Staphylococcus epidermidisbiofilm model. These systems could be effective in the prevention and treatment of topical infections. In another study, a molecularly related drug with an extremely poor water solubility, triclocarban (solubility¼50 ng/mL), was evaluated in poloxamines T1107 and T1307.32 Both copolymers display similar HLB, though T1307 solubilized the drug more efficiently due to a greater molecular weight and a larger micellar core.
To investigate the chemical stabilization of simvastatin, a hypolipidemic drug that in the stomach is reversibly converted from its absorbable lactonic form to an open carboxylic one (decreasing oral bioavailability), a broad spectrum of drug-loaded poloxamines was prepared.84 Some derivatives improved the solubility up to 152 times and partially or completely prevented the hydrolysis. In this work, the self-aggregation behavior of N-methylated poloxamine T1107 was assessed for the first time. This chemically modified derivative improved the encapsulation capacity and stability of simvastatin, strongly suggesting a greater drug–core interaction than the pristine derivative.
Our group has recently explored a broad variety of linear and branched PEO-PPOs for the encapsulation of different antiretrovirals employed in the treatment of the human immunodeficiency virus (HIV) infection. EFV is a first- line antiretroviral for the treatment of HIV-infected children above 3 years of age. EFV displays several (bio)pharmaceutic drawbacks such as poor aqueous solubility, low oral bioavailability and high inter- and intra-subject variability.
To improve the (bio)pharmaceutic properties of the drug and develop a pediatric formulation, EFV was encapsulated within single and mixed micelles.114–117 The aqueous solubility of the drug was increased more than 8400 times (from 4mg/mL to 34 mg/mL).39,114Regardless of the great drug payload, the size was usuallyo100 nm and morphology was spherical (Figure 5.1A).39,114Poloxamine T904 showed the best encapsulation capacity of all the investigated copolymers, though it was less physically stable than F127 micelles. Thus, aiming to improve both features F127/T904 mixed micelles were developed.117 Remarkably, EFV-loaded T904 and F127 micelles were physically stable under strong dilution (up to 1 : 75) over 1 month, at 371C.116 Extensive preclinical investigations of micelles with different composition and size, different drug payload and dose and administration conditions showed a statistically significant increase of the oral bioavailability with respect to a compounded suspension and an oily solution (Figure 5.1B–D).114,115 The administration of the copolymers did not show any acute adverse effect.
A remarkable advantage of poloxamers from a translational perspective over other experimental copolymers is that some of them have been approved as pharmaceutical excipients. Thus they can be used in clinical trials without further evaluations. On the other hand, it should be stressed that poloxamines display two main drawbacks: (i) a more limited variety of derivatives are commercially available and (ii) they are not currently approved as pharmaceutical excipients for the production of medicines. Thus, regardless of the greater encapsulation efficiency of poloxamine T904 with respect to F127, this later copolymer was chosen for the preparation of an EFV-loaded micellar system to be tested regarding oral bioavailability and compared to drug
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capsules, in a clinical trial with adult healthy volunteers. This versatile platform has also been implemented to investigate the interaction of EFV with the breast cancer resistant protein (BCRP) pump in intestine and central nervous system.118,119In this case, the administration route was oral or intravenous.
The blood-brain barrier (BBB) prevents the passage of xenobiotics from plasma into the central nervous system (CNS) and contributes to the generation of one of the most challenging HIV sanctuaries.120,121The presence of the virus and the limited access of antiretrovirals may result in HIV-1 encephalitis (HIVE-1), a disease that is more frequent in neonates and children.122–124To target EFV to the CNS without the need of expensive chemically modified nanocarriers, recently these EFV-loaded micelles were administered by intra- nasal route.125The CNS bioavailability and the relative exposure index (REI) increased four and five times, respectively, compared to the systems administered intravenously; REI was calculated as the ratio between the area-under-the-curve in CNS and in plasma. In a different application, the ionizable groups of poloxamine were capitalized to complex and stabilize negatively charged DNA,126 and also to transfect plasmid DNA in skeletal musclein vivo.127,128
0 2000 4000 6000 8000
0 4 8 12 16 20 24
Time (h) EFV plasma concentration (ng/mL)
Dose EFV: 20 mg/kg
0 400 800 1200
0 2 4 6 8
Time (h) EFV plasma concentration (ng/mL)
pF127 (10%) Suspension Triglyceride solution
0 2000 4000 6000 8000
0 4 8 12 16 20 24
Time (h) EFV plasma concentration (ng/mL)
pF127 (10%) Suspension Triglyceride solution
pF127 (10%) Suspension Triglyceride solution Dose EFV: 40 mg/kg
0 2000 4000 6000 8000
0 4 8 12 16 20 24
Time (h) EFV plasma concentration (ng/mL)
Dose EFV: 80 mg/kg
A B
C D
Figure 5.1 (A) Transmission-electron micrograph of EFV-loaded 10% micelles of poloxamine T1307 in buffer (pH 5.0) and negatively stained with 2%
phosphotungstic acid. Arrows point out the presence of spherical micelles of variable sizes. (B–D) EFV plasma concentrations after the oral administration of: (A) 20 mg/kg, (B) 40 mg/kg and (C) 80 mg/kg. Results are expressed as meanS.E. (n¼8).
(Reproduced from (A) Ref. 114 with permission of Future Medicine and (B–D) Ref. 115 with permission of Elsevier.)
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To overcome different drawbacks, such as the low viscosity of PEO-PPO gels that usually leads to short residence time at the application sitein vivo, different research groups employed poloxamers as platforms to design a variety of counterparts with improved features. Cohnet al.129–135 improved the macro- and microviscosity of the gels by means of chain extension andin situ cross- linking. The same research group introduced cross-linked thermo-responsive nanoshells with interesting features.136,137 Alvarez-Lorenzo et al.138 grafted poloxamers with poly(acrylic acid) segments to solubilize and chemically stabilize the lactonic form of the antitumoral drug camptothecin; the open carboxylic form is not active. The amphiphiles became dually responsive to temperature and pH. Interestingly, results suggested that the drug was solu- bilized by both the core and the corona. This aspect deserves a separate comment as it is usually assumed that only drug–core interactions govern the encapsulation capacity of polymeric micelles, but there are a few studies that demonstrated that also drug–corona interactions can contribute to increase the encapsulation capacity of the nanocarrier.31,138
Encapsulated drugs can modify the self-assembly of the copolymer. In other words, parameters such as CMC, CMT, aggregation number, cloud point, and micellar size and size distribution can undergo substantial changes in the presence of small drug molecules. It seems that this phenomenon would be more relevant in the case of drugs that hinder the aggregation process. On the other hand, drugs that promote aggregation could be capitalized to improve the properties of amphiphiles displaying poor or incomplete micellization tendency. Most of the research prioritized the study of drug solubilization and did not assess the effect of the drug on the amphiphile aggregation. This incomplete characterization may result in inconsistent and unpredictable data in vitroand in vivo. Tontosakiset al.139reported that small o-xylene concen- trations increase the aggregation of poloxamers. A similar effect was observed with phenol.140Conversely, urea hampered the aggregation of PluronicsP85, leading to the increase of CMC and CMT.141Naproxen and indomethacin did not change the CMC of PluronicsF127, but the size of the micelles and the aggregation numbers decreased sharply.142More recently, the pro-aggregation performance of EFV on different pristine and N-alkylated poloxamines has been also described.39
A main drawback of PEO-PPOs, regardless of their molecular features, is that owing to the relatively low hydrophobicity and amorphousness of PPO the self-assembly is incomplete. This phenomenon is more remarkable for more hydrophilic counterparts, at 251C. Aiming to increase the micellization tendency and reduce the CMC, the group of Attwood replaced PPO by more hydrophobic polyethers such as poly(butylene oxide) (PBO), poly(styrene oxide) (PSO) and poly(phenyl glycidyl ether) (PGO) and reported a prolific bibliography on the aggregation phenomena of various derivatives.143–157 Different molecular architectures such as A-B diblocks, and A-B-A and B- A-B triblocks, where A and B are the hydrophilic and the hydrophobic component respectively, led to the generation of a broad spectrum of derivatives with unique and improved properties. Due to a greater
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hydrophobicity, these micelles displayed greater solubilization ability and physical stability than PEO-PPOs. Another aspect that merits consideration is the relative hydrophobicity of the new segment with respect to PPO. For example, PBO is four times more hydrophobic than PPO;94 thus, 4-fold PEO/PBO ratios are demanded to attain micellization properties similar to those of PEO-PPO copolymers. In addition, as opposed to PPO that is 100%
amorphous, PBO, PSO and PGO can undergo crystallization. This difference may demand changes in the preparation procedure, more prolonged solubil- ization time at low temperature and additional studies to assess their physical stability in suspension and their ability to withstand lyophilization.153
In any event, the fact that PEO-PPOs are commercially available and that some of them have already been approved by the main regulatory agencies and that additional ones are under clinical evaluation constitutes a remarkable advantage over other experimental copolymers even if they display improved features.