International Journal of Pharmaceutics 462 (2014) 103–107
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Note
Interplay of stimuli-responsiveness, drug loading and release for a surface-engineered dendrimer delivery system Ruihong Liu a , Mingjing Sun b , Xipan Liu a , Aiping Fan a , Zheng Wang a,∗ , Yanjun Zhao a,∗ a Tianjin Key Laboratory for Modern Drug Delivery & High Efficiency, School of Pharmaceutical Science & Technology, Tianjin University, 92 Weijin Road, Tianjin 300072, China b College of Science, Tianjin University of Science & Technology, 29 TEDA 13th Avenue, Tianjin 300457, China
a r t i c l e
i n f o
Article history: Received 15 October 2013 Received in revised form 13 November 2013 Accepted 15 December 2013 Available online 25 December 2013 Keywords: Dendrimers Stimuli-responsive Dendrimer–drug interactions Surface-engineering Controlled release
a b s t r a c t The objectives of this study were to generate novel thermo and pH dual responsive poly(amidoamine) (PAMAM) via precise surface engineering, and investigate the interplay of dendrimer stimuliresponsiveness and the loading and release properties of a model agent, vitamin E acetate (VEAc). A higher dendrimer generation and maximized VEAc loading at elevated pH all contributed to a lower cloud point (CP) of the dendrimer–VEAc complex. The drug loading in G3.5 surface-engineered PAMAM was 22 mol/mol (pH 7.0) and 10 mol/mol (pH 5.0), which corresponded to a complex CP value at ca. 13 ◦ C (pH 7.0) and 46 ◦ C (pH 5.0), respectively. At physiological conditions, only less than 40% of VEAc was liberated when reaching the plateau, whilst more than 90% of VEAc was released from such system within 6 h at pH 5.0. This was due to the transition of dendrimer surface from dehydrated state to hydrated state upon pH dropping, enabling rapid drug release for therapeutic action. This smart stimuli-responsive dendritic delivery system holds promise for the efficient drug delivery to tissues with pH abnormality such as tumor. © 2013 Elsevier B.V. All rights reserved.
Dendrimers are highly branched and symmetrical macromolecules with specific chemical composition, precise molecular weight, and well-defined core-interior-periphery architecture that distinguish them from traditional polydisperse linear polymers (Buhleier et al., 1978). The size, shape, topology, and multivalency of dendrimers are determined by the generation (G); at high-generation, dendrimers are relatively larger, exhibiting a globular morphology with more surface terminal groups (Tomalia, 1991). The quantity, type, and characteristics of surface terminal groups govern the properties of dendrimers such as aqueous solubility, biocompatibility, and multifunctionality in terms of biomedical and pharmaceutical applications (Shi et al., 2007). The empty interior cavity of dendrimers could be used for physical encapsulation of different guest molecules or small particles (Astruc et al., 2010). Taking advantage of abundant terminal groups, various ligands can be conjugated to the dendrimer periphery via surface engineering, including therapeutic agents, imaging molecules, targeting fragments and other functional moieties.
∗ Corresponding authors at: School of Pharmaceutical Science & Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, China. Tel.: +86 22 2740 4018; fax: +86 22 2740 4018. E-mail addresses: [email protected] (Z. Wang), [email protected] (Y. Zhao). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.12.031
These smart features enable the application of dendrimers in drug delivery and targeting, medical diagnosis, tissue engineering, and biosensor (Lee et al., 2005). Tailored surface engineering was attractively employed to produce smart dendrimers with thermosensitive or other stimuliresponsive properties for the reduction of side effects and maximum manifestation of guest molecule action (Kojima, 2010; Kono et al., 2011; Mura et al., 2013). Both thermosensitive linear polymers (e.g. poly(N-isopropylacrylamide)) and functional small molecules have been employed for generating a temperaturesensitive dendrimer shell (Haba et al., 2004; You et al., 2004; Haba et al., 2007). However, the use of polymers for surface modification would lose the molecular uniformity of dendrimers due to the inherently polydisperse nature of linear polymers. From this point of view, small molecules are preferred when designing thermosensitive dendrimers. High generation dendrimers present more surface groups for multi-functionalization, but concerns over their cytotoxicity and hemolysis has been raised regarding the pharmaceutical applications (Duncan and Izzo, 2005). The thermosensitivity of surface-engineered dendrimers was often indicated by a parameter named “the lower cloud point (CP)”. The nature and density of the terminal functional groups control the extent of hydrophobic interaction among these groups and thus the level of conformational switch of dendrimer periphery between hydrated and dehydrated states (Tono et al., 2006). The hydrophobicity of dendrimer interior also affects the thermosensitivity since
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the dendrimer is an integrated unity of interior and periphery. Hence the encapsulation of active pharmaceutical ingredients (API) in the dendrimer interior would theoretically make an impact on the thermosensitivity of dendrimer–API complexes; hydrophilic guests would increase the CP value, whereas hydrophobic ones led to the opposite way. Poly(amidoamine) (PAMAM) is one of the mostly investigated dendrimers because of the superior adaptability of thermoresponsiveness with primary amine, hydroxyl, and carboxyl terminal groups available for surface modification (Zhao et al., 2011). The existence of the ionizable tertiary amine groups in the building blocks also endows the PAMAM dendrimer intrinsic acid-responsiveness. Therefore, surface-modified dual pH- and temperature-sensitive PAMAM dendrimers could be precisely engineered to exhibit a CP value around physiologically relevant temperature (37–42 ◦ C) and pH (5.0–7.4), which holds promise for controlled drug delivery at the target sites (Jin et al., 2011; Li et al., 2013). The majority of previous investigations focused on the screening and optimization of desired terminal functional groups or their combination to accurately regulate the thermoresponsiveness of dendrimers, while the role of guest molecules (i.e. API) in these processes were usually neglected. The current study aimed to figure out the extent to which the host–guest interaction influences the thermosensitivity of
Scheme 1. The schematic structure of surface-engineered thermosensitive PAMAMTris -DOA.
Fig. 1. The thermosensitivity of two generations (G) of surface-modified poly(amidoamine) (PAMAM) dendrimers with or without the model guest molecule (vitamin E acetate/VEAc): (A) placebo thermosensitive G2.5 PAMAM; (B) placebo thermosensitive G3.5 PAMAM; (C) VEAc-loaded thermosensitive G2.5 PAMAM; (D) VEAc-loaded thermosensitive G3.5 PAMAM. Data were presented as the transmittance at 500 nm against temperature under differing aqueous buffer conditions from pH 9.0 to pH 4.0.
R. Liu et al. / International Journal of Pharmaceutics 462 (2014) 103–107
105
Fig. 2. Maximum loading of guest molecule (vitamin E acetate) in thermosensitive (G2.5 and G3.5) PAMAMTris -DOA dendrimer (A); data were presented as the molar ratio of guest and dendrimer (mol/mol) at pH (4–9) (n = 3). Effect of dendrimer binding with varying equivalents (equiv.) of guest molecule on the thermosensitivity of G2.5 (B) and G 3.5 (D) PAMAMTris -DOA; the aqueous medium pH was set at 7.0. The release profiles of guest molecule from G3.5 PAMAMTris -DOA dendrimer at pH 7.4 and 5.0 (n = 3) (C); the temperature was 37 ◦ C that was above the cloud point of dendrimer at pH 7.4, but below the cloud point of dendrimer at pH 5.0.
the dendrimer–API complexes, and the corresponding impact on the controlled release of API under physiological circumstances. As most active pharmaceutical ingredients are hydrophobic, a hydrophobic agent, vitamin E acetate (VEAc) was selected as the model guest molecule in the current study. Based on our previous work, 4-(diethylamino)-4-oxobutanoic acid (DOA) was selected for surface engineering of Tris-modified low generation PAMAM (G2.5 or G3.5) dendrimers (abbreviated as PAMAMTris -DOA, Scheme 1) because the CP values of such dendrimers were found close to the normal human body temperature (Zhao et al., 2012). Firstly, we investigated the effect of maximum API loading on the thermosensitivity of two types of PAMAMTris -DOA (G2.5 and G3.5) at different pH (Fig. 1). Due to the presence of the tertiary amines in the interior and exterior (i.e. DOA) of dendrimers, PAMAMTris -DOA showed pH-dependent temperature sensitivity irrespective of the generation and guest loading; the CP value at low pH was larger in contrast to that at high pH. This was mainly a consequence of differing ionization state of tertiary amines (pKa ∼ 6.5) at different pH (D’Emanuele and Attwood, 2005). A typical example was that placebo G2.5 PAMAMTris -DOA showed no thermosensitivity at pH 4.0, which was a result of the high degree of dendrimer ionization and somewhat low compactness of surface DOA (Fig. 1A). However, placebo G3.5 dendrimer exhibited thermosensitivity at pH 4.0 simply owing to the increased surface density of DOA, facilitating their hydrophobic interaction and hence dehydration at elevated temperature (Fig. 1B). Interestingly, VEAc loading also enabled G2.5 dendrimer thermosensitive at pH 4.0 for the reason that the inclusion of hydrophobic VEAc increased the
hydrophobicity of the whole dendrimer–API complex (Fig. 1C). As the DOA molecule contains both alkane group (for hydrophobic interaction) and tertiary amine group, it is the interplay of medium pH and surface density of DOA that determines the thermosensitivity of placebo PAMAMTris -DOA. Increasing the dendrimer generation was an effective approach to control surface compactness of DOA. The use of Tris-modified PAMAM further amplified such effect (Zhao et al., 2012). The inclusion of VEAc further complicates the phase transition behavior of the dendrimer–guest complex. At different pH, the interior tertiary amine of the dendrimer exhibited dissimilar ionization state. At lower pH (4–5), the dendrimer cavity is highly ionized (> 90%) and hydrophilic, leading to weak affinity with the hydrophobic guest and thus poor loading (Fig. 2A). In contrast, the hydrophobic internal cavity at high pH (8–9) as a consequence of the low degree of ionization (
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Note
Interplay of stimuli-responsiveness, drug loading and release for a surface-engineered dendrimer delivery system Ruihong Liu a , Mingjing Sun b , Xipan Liu a , Aiping Fan a , Zheng Wang a,∗ , Yanjun Zhao a,∗ a Tianjin Key Laboratory for Modern Drug Delivery & High Efficiency, School of Pharmaceutical Science & Technology, Tianjin University, 92 Weijin Road, Tianjin 300072, China b College of Science, Tianjin University of Science & Technology, 29 TEDA 13th Avenue, Tianjin 300457, China
a r t i c l e
i n f o
Article history: Received 15 October 2013 Received in revised form 13 November 2013 Accepted 15 December 2013 Available online 25 December 2013 Keywords: Dendrimers Stimuli-responsive Dendrimer–drug interactions Surface-engineering Controlled release
a b s t r a c t The objectives of this study were to generate novel thermo and pH dual responsive poly(amidoamine) (PAMAM) via precise surface engineering, and investigate the interplay of dendrimer stimuliresponsiveness and the loading and release properties of a model agent, vitamin E acetate (VEAc). A higher dendrimer generation and maximized VEAc loading at elevated pH all contributed to a lower cloud point (CP) of the dendrimer–VEAc complex. The drug loading in G3.5 surface-engineered PAMAM was 22 mol/mol (pH 7.0) and 10 mol/mol (pH 5.0), which corresponded to a complex CP value at ca. 13 ◦ C (pH 7.0) and 46 ◦ C (pH 5.0), respectively. At physiological conditions, only less than 40% of VEAc was liberated when reaching the plateau, whilst more than 90% of VEAc was released from such system within 6 h at pH 5.0. This was due to the transition of dendrimer surface from dehydrated state to hydrated state upon pH dropping, enabling rapid drug release for therapeutic action. This smart stimuli-responsive dendritic delivery system holds promise for the efficient drug delivery to tissues with pH abnormality such as tumor. © 2013 Elsevier B.V. All rights reserved.
Dendrimers are highly branched and symmetrical macromolecules with specific chemical composition, precise molecular weight, and well-defined core-interior-periphery architecture that distinguish them from traditional polydisperse linear polymers (Buhleier et al., 1978). The size, shape, topology, and multivalency of dendrimers are determined by the generation (G); at high-generation, dendrimers are relatively larger, exhibiting a globular morphology with more surface terminal groups (Tomalia, 1991). The quantity, type, and characteristics of surface terminal groups govern the properties of dendrimers such as aqueous solubility, biocompatibility, and multifunctionality in terms of biomedical and pharmaceutical applications (Shi et al., 2007). The empty interior cavity of dendrimers could be used for physical encapsulation of different guest molecules or small particles (Astruc et al., 2010). Taking advantage of abundant terminal groups, various ligands can be conjugated to the dendrimer periphery via surface engineering, including therapeutic agents, imaging molecules, targeting fragments and other functional moieties.
∗ Corresponding authors at: School of Pharmaceutical Science & Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, China. Tel.: +86 22 2740 4018; fax: +86 22 2740 4018. E-mail addresses: [email protected] (Z. Wang), [email protected] (Y. Zhao). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.12.031
These smart features enable the application of dendrimers in drug delivery and targeting, medical diagnosis, tissue engineering, and biosensor (Lee et al., 2005). Tailored surface engineering was attractively employed to produce smart dendrimers with thermosensitive or other stimuliresponsive properties for the reduction of side effects and maximum manifestation of guest molecule action (Kojima, 2010; Kono et al., 2011; Mura et al., 2013). Both thermosensitive linear polymers (e.g. poly(N-isopropylacrylamide)) and functional small molecules have been employed for generating a temperaturesensitive dendrimer shell (Haba et al., 2004; You et al., 2004; Haba et al., 2007). However, the use of polymers for surface modification would lose the molecular uniformity of dendrimers due to the inherently polydisperse nature of linear polymers. From this point of view, small molecules are preferred when designing thermosensitive dendrimers. High generation dendrimers present more surface groups for multi-functionalization, but concerns over their cytotoxicity and hemolysis has been raised regarding the pharmaceutical applications (Duncan and Izzo, 2005). The thermosensitivity of surface-engineered dendrimers was often indicated by a parameter named “the lower cloud point (CP)”. The nature and density of the terminal functional groups control the extent of hydrophobic interaction among these groups and thus the level of conformational switch of dendrimer periphery between hydrated and dehydrated states (Tono et al., 2006). The hydrophobicity of dendrimer interior also affects the thermosensitivity since
104
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the dendrimer is an integrated unity of interior and periphery. Hence the encapsulation of active pharmaceutical ingredients (API) in the dendrimer interior would theoretically make an impact on the thermosensitivity of dendrimer–API complexes; hydrophilic guests would increase the CP value, whereas hydrophobic ones led to the opposite way. Poly(amidoamine) (PAMAM) is one of the mostly investigated dendrimers because of the superior adaptability of thermoresponsiveness with primary amine, hydroxyl, and carboxyl terminal groups available for surface modification (Zhao et al., 2011). The existence of the ionizable tertiary amine groups in the building blocks also endows the PAMAM dendrimer intrinsic acid-responsiveness. Therefore, surface-modified dual pH- and temperature-sensitive PAMAM dendrimers could be precisely engineered to exhibit a CP value around physiologically relevant temperature (37–42 ◦ C) and pH (5.0–7.4), which holds promise for controlled drug delivery at the target sites (Jin et al., 2011; Li et al., 2013). The majority of previous investigations focused on the screening and optimization of desired terminal functional groups or their combination to accurately regulate the thermoresponsiveness of dendrimers, while the role of guest molecules (i.e. API) in these processes were usually neglected. The current study aimed to figure out the extent to which the host–guest interaction influences the thermosensitivity of
Scheme 1. The schematic structure of surface-engineered thermosensitive PAMAMTris -DOA.
Fig. 1. The thermosensitivity of two generations (G) of surface-modified poly(amidoamine) (PAMAM) dendrimers with or without the model guest molecule (vitamin E acetate/VEAc): (A) placebo thermosensitive G2.5 PAMAM; (B) placebo thermosensitive G3.5 PAMAM; (C) VEAc-loaded thermosensitive G2.5 PAMAM; (D) VEAc-loaded thermosensitive G3.5 PAMAM. Data were presented as the transmittance at 500 nm against temperature under differing aqueous buffer conditions from pH 9.0 to pH 4.0.
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Fig. 2. Maximum loading of guest molecule (vitamin E acetate) in thermosensitive (G2.5 and G3.5) PAMAMTris -DOA dendrimer (A); data were presented as the molar ratio of guest and dendrimer (mol/mol) at pH (4–9) (n = 3). Effect of dendrimer binding with varying equivalents (equiv.) of guest molecule on the thermosensitivity of G2.5 (B) and G 3.5 (D) PAMAMTris -DOA; the aqueous medium pH was set at 7.0. The release profiles of guest molecule from G3.5 PAMAMTris -DOA dendrimer at pH 7.4 and 5.0 (n = 3) (C); the temperature was 37 ◦ C that was above the cloud point of dendrimer at pH 7.4, but below the cloud point of dendrimer at pH 5.0.
the dendrimer–API complexes, and the corresponding impact on the controlled release of API under physiological circumstances. As most active pharmaceutical ingredients are hydrophobic, a hydrophobic agent, vitamin E acetate (VEAc) was selected as the model guest molecule in the current study. Based on our previous work, 4-(diethylamino)-4-oxobutanoic acid (DOA) was selected for surface engineering of Tris-modified low generation PAMAM (G2.5 or G3.5) dendrimers (abbreviated as PAMAMTris -DOA, Scheme 1) because the CP values of such dendrimers were found close to the normal human body temperature (Zhao et al., 2012). Firstly, we investigated the effect of maximum API loading on the thermosensitivity of two types of PAMAMTris -DOA (G2.5 and G3.5) at different pH (Fig. 1). Due to the presence of the tertiary amines in the interior and exterior (i.e. DOA) of dendrimers, PAMAMTris -DOA showed pH-dependent temperature sensitivity irrespective of the generation and guest loading; the CP value at low pH was larger in contrast to that at high pH. This was mainly a consequence of differing ionization state of tertiary amines (pKa ∼ 6.5) at different pH (D’Emanuele and Attwood, 2005). A typical example was that placebo G2.5 PAMAMTris -DOA showed no thermosensitivity at pH 4.0, which was a result of the high degree of dendrimer ionization and somewhat low compactness of surface DOA (Fig. 1A). However, placebo G3.5 dendrimer exhibited thermosensitivity at pH 4.0 simply owing to the increased surface density of DOA, facilitating their hydrophobic interaction and hence dehydration at elevated temperature (Fig. 1B). Interestingly, VEAc loading also enabled G2.5 dendrimer thermosensitive at pH 4.0 for the reason that the inclusion of hydrophobic VEAc increased the
hydrophobicity of the whole dendrimer–API complex (Fig. 1C). As the DOA molecule contains both alkane group (for hydrophobic interaction) and tertiary amine group, it is the interplay of medium pH and surface density of DOA that determines the thermosensitivity of placebo PAMAMTris -DOA. Increasing the dendrimer generation was an effective approach to control surface compactness of DOA. The use of Tris-modified PAMAM further amplified such effect (Zhao et al., 2012). The inclusion of VEAc further complicates the phase transition behavior of the dendrimer–guest complex. At different pH, the interior tertiary amine of the dendrimer exhibited dissimilar ionization state. At lower pH (4–5), the dendrimer cavity is highly ionized (> 90%) and hydrophilic, leading to weak affinity with the hydrophobic guest and thus poor loading (Fig. 2A). In contrast, the hydrophobic internal cavity at high pH (8–9) as a consequence of the low degree of ionization (
